Method for measuring polarization of bathochromically shifted fluorescence

ABSTRACT

An improved method for measuring polarized fluorescence emissions compensates for background emissions without separating a fluorescing material from background material contributing background fluorescence. The method is particularly useful in measuring fluorescence from a suspension of cells such as stimulated lymphocytes in the SCM assay, where the intracellular fluorescence is due to the penetration of the cells by a fluorogenic agent precursor and its subsequent hydrolysis. The method involves measuring the horizontally and vertically polarized components of the fluorescence emission at a primary wavelength and at least one secondary wavelength. The secondary wavelength is selected based on the bathochromic shift of the spectrum of the fluorescence emissions from the fluorescing material as compared to the fluorescence emissions from the background. From these measurements a factor representing the fraction of the total intensity of fluorescence emission due to background fluorescence at the primary wavelength is determined, from which the intensities of the vertically and horizontally polarized fluorescence emissions due to background fluorescence are derived. These derived background intensities are then subtracted from the vertically and horizontally polarized fluorescence emission intensities measured at the primary wavelength to obtain intensities due solely to the material being analyzed. More than one secondary wavelength can be used to increase the accuracy of the method. Apparatus suitable for practicing this method is described.

CROSS-REFERENCE

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 867,079, entitled "Method for Measuring PolarizedFluorescence Emissions," filed May 27, 1986, now abandoned, andincorporated herein by this reference.

BACKGROUND

Many diseases occurring in humans and animals can be detected by thepresence of foreign substances, particularly in the blood, which arespecifically associated with the disease or condition. Tests forantigens or other such substances produced as a result of such diseasesshow great promise as a diagnostic tool for the early detection andtreatment of the particular disease that produced the antigen or othersubstance. Procedures for the detection of such substances must bereliable, reproducible, and sensitive in order to constitute a practicaldiagnostic procedure for health care providers. In addition, any suchprocedure should be able to be carried out by persons of ordinary skilland training in laboratory procedure, and should be relatively fast andinexpensive. Preferably, such procedures should be readily adaptable toinstrumentation and automation, as required if such procedures are to becarried out on a large scale.

For example, in the treatment of the various malignancies that afflicthumans and animals, referred to generally as cancer, it is recognizedthat early detection is a key to effective treatment, especially as manytherapeutic procedures are effective only in relatively early stages ofthe disease. In fact, virtually all known cancer treatments are not onlymore effective, but safer, when administered in early stages of cancer.Far too many cases of cancer are only discovered too late for effectivetreatment.

Accordingly, there is a great need for rapid, easy-to-perform, andreliable tests which can diagnose cancer at early stages. In thisconnection new tests and procedures are being developed to effect earlydiagnosis of the cancer.

We have developed and reported one such test for the early detection ofcancer in L. Cercek, B. Cercek, and C.I.V. Franklin, "BiophysicalDifferentiation Between Lymphocytes from Healthy Donors, Patients withMalignant Diseases and Other Disorders," Brit. J. Cancer 29, 245-3521974) and L. Cercek and B. Cercek, "Application of the Phenomenon ofChanges in the Structuredness of Cytoplasmic Matrix (SCM) in theDiagnosis of Malignant Disorder: a Review," Europ. J. Cancer 13, 903-915(1977), which are incorporated herein by this reference.

Our basic SCM test includes the steps of:

(1) challenging a selected subpopulation of lymphocytes from a donorwith a challenging agent such as a mitogen or an antigen associated witha condition or disease, such as cancer; and

(2) determining the change in structuredness of the cytoplasmic matrix(SCM) of the challenged lymphocytes, typically using fluorescencepolarization.

When applied to cancer, our SCM (structuredness of cytoplasmic matrix)test is based on the phenomenon that the internal structure of aselected subpopulation of the lymphocytes from a healthy individual isaltered when challenged by a mitogen such as phytohaemagglutinin (PHA)but is not altered by other selected challenging agents, such as certaincancerassociated antigens. Contrarily, the equivalent subpopulation oflymphocytes from an individual with cancer responds oppositely. In otherwords the same subpopulation of lymphocytes from cancer patients doesnot respond in the SCM test when challenged by a mitogen, but doesrespond strongly to challenge by a number of cancer-associated antigens.

The change seen in SCM are believed to reflect changes in the internalstructure of the lymphocyte as the lymphocyte is activated forsynthesis. Similar changes can occur in living cells other thanlymphocytes during the cell cycle and growth of the cells. Such changescan also be evoked by various external agents, such as ionizingradiation, mechanical forces, chemicals, growth inhibiting andstimulating agents, etc. These changes can be conveniently monitoredwith a specially adapted technique of fluorescein fluorescencepolarization, as we have published in numerous articles, including L.Cercek and B. Cercek, "Studies on the Structuredness of Cytoplasm andRates of Enzymatic Hydrolysis in Growing Yeast Cells. I. Changes Inducedby Ionizing Radiation," Int. J. Radiat. Biol. 21, 445-453 (1972); L.Cercek and B Cercek, "Studies on the Structuredness of Cytoplasm andRates of Enzymatic Hydrolysis in Growing Yeast Cells. II. ChangesInduced by Ultra-Violet Light," Int. J. Radiat. Biol 22. 539-544 (1972);L. Cercek and B. Cercek, "Relationship Between Changes in theStructuredness of Cytoplasm and Rate Constants for the Hydrolysis of FDAin Saccharomyces cerevisiae," Biophysik 9, 109-112 (1973); L. Cercek, B.Cercek, and C. H. Ockey, "Structuredness of the Cytoplasmic Matrix andMichaelis-Menten Constants for the Hydrolysis of FDA During the CellCycle in Chinese Hamster Ovary Cells," Biophysik 10 187-194 (1973) B. I.Lord, L. Cercek, B. Cercek, G. P. Shah, T. M. Dexter and L. G. Lajtha,"Inhibitors of Haemopoietic Cell Proliferation: Specificity of ActionWithin the Haemopoietic System," Brit. J. Cancer 29 168-175 (1974); L.Cercek and B. Cercek, "Involvement of Cyclic-AMP in Changes of theStructuredness of Cytoplasmic Matrix," Radiat. & Environ. Biophys. 11,209-212 (1974); L. Cercek, P. Milenkovic, B. Cercek, & L. G. Lajtha,"Induction of PHA Response in Mouse Bone Marrow Cells by Thymic Extractsas Studied by Changes in the Structuredness of Cytoplasmic Matrix,"Immunology 29, 885-891 (1975); L. Cercek and B. Cercek, "Effects ofOsmomolarity, Calcium and Magnesium Ions on the Structuredness ofCytoplasmic Matrix (SCM)," Radiat. & Environ. Biophys. 13, 9-12 (1976);L. Cercek & B. Cercek, "Changes in the Structuredness of CytoplasmicMatrix (SCM) Induced in Mixed Lymphocyte Reactions," Radiat. & Environ.Biophys. 13, 71-74 (1976); L. Cercek, B. Cercek, & C. H. Ockey,"Fluorescein Excitation and Emission Polarization Spectra in LivingCells: Changes During the Cell Cycle," Biophys. J. 23, 395-405 (1978) L.Cercek and B. Cercek, "Involvement of Mitochondria in Changes ofFluorescein Excitation and Emission Polarization Spectra in LivingCells," Biophys. J. 28, 403-412 (1979); L. Cercek, B. Cercek, and B. I.Lord, "The Effect of Specific Growth Inhibitors on FluoresceinFluorescence Polarization Spectra in Haemopoietic Cells," Brit. J.Cancer, 44 749-752 (1981); and L. Cercek and B. Cercek, "Effects ofAscorbate Ions on Intracellular Fluorescein Emission PolarizationSpectra in Cancer and Normal Proliferating Cells," Cancer Detection andPrevention 10, 1-20 (1987), all of which are incorporated herein by thisreference.

The usefulness of this SCM test for the detection of cancer has beendocumented in numerous articles. Articles from our laboratory include:L. Cercek, B. Cercek, and J. V. Garrett, "Biophysical DifferentiationBetween Normal Human and Chronic Lymphocytic Leukaemia Lymphocytes," inLymphocyte Recognition and Effector Mechanisms (K. Lindahl-Kiessling andD. Osoba eds., New York, Academic Press, 1974), pp. 553-558; L. Cercek,B. Cercek and C. I. V. Franklin, "Biophysical Differentiation betweenLymphocytes from Healthy Donors, Patients with Malignant Disease andOther Disorders," Brit. J. Cancer 29 345-352 (1974); L. Cercek and B.Cercek, "Changes in the SCM Response Ratio (RR_(SCM)) After SurgicalRemoval of Malignant Tissue," Brit. J. Cancer 31, 250-251 (1975); L.Cercek and B. Cercek, "Apparent Tumour Specificity with the SCM Test,"Brit. J. Cancer 31, 252-253 (1975); L. Cercek and B. Cercek, "Changes inthe Structuredness of Cytoplasmic Matrix of Lymphocytes as a Diagnosticand Prognostic Test for Cancer," in Cell Biology and Tumour Immunology,Excerpta Medica International Congress Series No. 349, Proceedings ofthe XI International Cancer Congress, Florence, 1974 (Amsterdam,Excerpta Medica, 1974), vol. 1, pp. 318-323) L. Cercek and B. Cercek,"Application of the Phenomenon of Changes in the Structuredness ofCytoplasmic Matrix (SCM) in the Diagnosis of Malignant Disorders: aReview," Europ. J. Cancer 13, 903-915 (1977); L. Cercek and B. Cercek,"Detection of Malignant Diseases by Changes in the Structuredness ofCytoplasmic Matrix of Lymphocytes Induced by Phytohaemagglutinin andCancer Basic Proteins," in Tumour Markers, Determination and ClinicalRole: Proceedings of the Sixth Tenovus Workshop, Cardiff, April 1977 (K.Griffith, A. M. Neville, and C. G. Pierrepoint, eds., Cardiff, AlphaOmega Publishing Co., 1978), pp. 215-226; and L. Cercek and B. Cercek,"Changes in SCM-Responses of Lymphocytes in Mice After Implantation withEhrlich Ascites Cells," Europ. J. Cancer 17, 167-171 (1981), all ofwhich are incorporated herein by this reference.

The usefulness of the SCM test has been confirmed in articles from otherlaboratories, including F. Takaku, K. Yamanaka, and Y. Hashimoto,"Usefulness of the SCM Test in the Diagnosis of Gastric Cancer," Brit.J. Cancer 36, 810-813 (1977); H. Kreutzmann, T. M. Fliedner, H. J.Galla, and E. Sackmann, "FluorescencePolarization Changes in MononuclearBlood Leucocytes After PHA Incubation: Differences in Cells fromPatients with and Without Neoplasia," Brit. J. Cancer 37, 797-805(1978); Y. Hashimoto, T. Yamanaka, and F. Takaku, "DifferentiationBetween Patients with Malignant Diseases and Non-Malignant Diseases orHealthy Donors by Changes of Fluorescence Polarization in the Cytoplasmof Circulating Lymphocytes," Gann 69, 145-149 (1978); J. A. V. Pritchardand W. H. Sutherland, "Lymphocyte Response to Antigen Stimulation asMeasured by Fluorescence Polarization (SCM-Test)," Brit. J. Cancer 38,339-343 (1978); J. A. V. Pritchard, J. E. Seaman, I. H. Evans, K. W.James, W. H. Sutherland, T. J. Deeley, I. J. Kerby, I. C. M. Patterson,and B. H. Davies, "Cancer-Specific Density Changes in LymphocytesFollowing Stimulation with Phytohaemagglutinin," Lancet 11, 1275-1277(Dec. 16. 1978); H. Orjasaeter, G. Jordfald, and I. Svendsen, "Responseof T-Lymphocytes to Phytohaemagglutinin (PHA) and toCancer-Tissue-Associated Antigens, Measured by the IntracellularFluorescence Folarization Technique (SCM Test)," Brit. J. Cancer 40,628-633 (1979); N. D. Schnuda, "Evaluation of Fluorescence Polarizationof Human Blood Lymphocytes (SCM Test) in the Diagnosis of Cancer,"Cancer 46, 1164-1173 (1980); J. A. V. Pritchard, W. H. Sutherland, J. E.Siddall, A. J. Bater, I. J. Kerby, T. J. Deeley, G. Griffith, R.Sinclair, B. H. Davies, A. Rimmer, & D. J. T. Webster, "A ClinicalAssessment of Fluorescence Polarisation Changes in LymphocytesStimulated by Phytohaemagglutinin (PHA) in Malignant and BenignDisease," Europ. J. Cancer, Clin. Oncol. 18, 651-659 (1982); G. R.Hocking, J. M. Rolland, R. C. Nairn, E. Pihl, A. M. Cuthbertson, E. S.R. Hughes, and W. R. Johnson, "Lymphocyte Fluorescence PolarizationChanges After Phytohaemagglutinin Stimulation in the Diagnosis ofColorectal Carcinoma," J. National Cancer Inst. 68 579-583 (1982); M.Deutsch and A. Weinreb, "Validation of the SCM-Test for the Diagnosis ofCancer," Eur. J. Cancer, Clin. Oncol. 19, 187-193 (1983); S. Chaitchik,O. Asher, M. Deutsch. and A. Weinreb, "Tumour Specificity of the SCMTest for Cancer Diagnosis," Europ. J. Cancer, Clin. Oncol. 21 1165-1170(1985); and J. Matsumoto, T. Tenzaki and T. Ishiguro, "ClinicalEvaluation of Fluorescein Polarization of Peripheral Lymphocytes (SCMTest) in the Diagnosis of Cancer," J. Japan Soc. Cancer Ther. 20,728-734 (1985), all of which are incorporated herein by this reference.

The SCM test can be applied to detection of diseases and conditionsother than cancer, such as viral and bacterial infections, determinationof allergic reactions, tissue typing, and monitoring of allograftrejections based on the SCM responses in mixed lymphocyte reactions, asdisclosed in the 1976 Radiation and Environmental Biophysics article byL. Cercek and B. Cercek. This extension of the SCM test is disclosed andclaimed in our co-pending U.S. patent application, Ser. No. 838,264,filed Mar. 10, 1986, now abandoned, and entitled "Separation and Use ofDensity Specific Blood Cells," which is incorporated herein by thisreference. The presence of other antigen-producing diseases and bodilyconditions does not interfere with the SCM test; a patient afflictedwith more than one type of antigen-producing disease can be tested for amultiplicity of such diseases simply by running separate tests using foreach test an antigen derived from each separate disease or conditionbeing tested for.

When fluorescence polarization is used to determine changes of SCM, suchchanges are seen as a decrease in the fluorescence polarization of thecells when polarized light is used to excite an extrinsic fluorgenerated intracellularly by the hydrolysis of a nonfluorescent compoundwhich has been absorbed by the lymphocytes. The fluor typically isfluorescein and the nonfluorescent compound is typically fluoresceindiacetate (FDA). The FDA serves as a fluorogenic agent precursor. Anextrinsic fluor is used because the intrinsic fluorescence of cellularcomponents is too small to give meaningful results in this test.Therefore, all references to fluorescence polarization values herein arereferences to fluorescence polarization values obtained with anextrinsic fluor, preferably one generated by enzymatic hydrolysis from anonfluorogenic compound added to and absorbed by the cells.

Fluorescence polarization is a measure of intracellular rigidity; thegreater the intracellular mobility, the less the measured fluorescencepolarization. As seen in the SCM test, the observed decrease influorescence polarization is believed to result mainly from changes inthe conformation of the mitochondria, the energy-producing organelles ofthe cell. The changes in the mitochondria are believed to result fromthe contractions of the cristae or inner folds of the mitochondrialmembrane. The SCM reflects the forces of interaction betweenmacromolecules and small molecules such as water molecules, ions,adenosine triphosphate, and cyclic adenosine monophosphate.Perturbations of these interactions result in changes in the SCM.

In our SCM test, the best indication of structuredness is not theabsolute fluorescence polarization measured, but rather the netfluorescence polarization (P). P is determined after correction is madefor: (i) intrinsic fluorescence of the medium in which the cells aresuspended; (ii) extracellular fluor present whether generated by leakageof fluor from cells or non-enzymatic hydrolysis of fluorescein diacetatein the medium; and (iii) unequal transmission of the two components ofpolarized light in the fluorescence polarization measurement apparatus.Thus all references to fluorescence polarization below are to netfluorescence polarization, P, unless indicated otherwise. When thefluorescence polarization measurements are performed on living cells, Pis the net intracellular polarization.

In our test, fluorescein is introduced into the cells by intracellularhydrolysis of the non-fluorogenic compound fluorescein diacetate whichhas been taken up by the lymphocytes. Then are measured the horizontallyand vertically polarized components of emitted fluorescence due toexcitation of the cell suspension by light from a suitable source, suchas vertically polarized blue light from a xenon lamp. The intensities ofthe vertically and horizontally polarized fluorescence components areused to calculate P. Challenged lymphocytes from a donor afflicted witha disease or condition associated with the challenging antigen exhibit asubstantial decrease of at least 10 percent in the fluorescencepolarization value, P, compared to non-challenged lymphocytes from thesame donor. On the other hand, challenged lymphocytes from donors notafflicted with the antigen-producing disease or condition do not exhibita significant decrease in P after contact with the challenging antigen.

As previously stated, the calculation of P includes corrections forseveral factors, including background fluorescence, in order to yieldmeaningful fluorescence polarization values. Ideally, since the FDA orother fluorogenic agent precursor itself does not fluoresce and is onlyconverted into a fluorescent compound such as fluorescein onintracellular hydrolysis by the lymphocytes, the background isrelatively small and consists of only the fluorescence resulting fromthe background material.

In practice, however, compensating for background fluorescence createsserious problems with the SCM measurements. As soon as the intracellularhydrolysis of FDA to fluorescein begins, some of the fluoresceinmolecules produced by the hydrolysis leak out of the cell and add tobackground fluorescence. Additionally, FDA is susceptible tononenzymatic or thermal hydrolysis resulting in still more fluoresceinpresent outside of the cell and a higher background. This backgroundsteadily increases as the fluorescence polarization is measured.

In our prior work, we compensated for this extracellular fluorescencebackground by filtering the lymphocyte suspension. Filtration was begunabout four to seven minutes after the recording of polarizedfluorescence intensities had begun. The vertically and horizontallypolarized components of the fluorescence emissions from the cell-freefiltrate as well as the length of time of the filtration step had to berecorded. The fluorescence polarization intensity measurements performedon the lymphocyte suspension before filtering then had to beextrapolated to the time point at which the filtration step was one-halfcompleted, and the fluorescence polarization measurements on thefiltrate then subtracted from the extrapolated measurements to obtainthe net intracellular vertically and horizontally polarized fluorescenceintensities due to the lymphocytes themselves. This measurement processis described in our 1977 European Journal of Cancer article.

In unskilled hands, the filtration step can introduce errors anduncertainties into the SCM results. For example, delay in filtration canintroduce uncertainties in the values of the extrapolated fluorescencepolarization measurements, since the intensity increases with time. Inaddition, if excess pressure is applied to the cells during filtration,the filtration step itself can damage cell membranes, resulting inleakage of intracellular fluorescein and fluoresceindiacetate-hydrolyzing enzyme into the filtrate. This leakage can causean artificially high background measurement. Not only can thefluorescein leaked into the filtrate directly increase the backgroundfluorescence, but the presence of fluorescein diacetate-hydrolyzingenzyme in the filtrate can convert some of the nonfluorescingfluorescein diacetate into fluorescein, further adding to thebackground.

Clinical consequences of an erroneously high background measurement canbe serious. Because fluorescence polarization is a measure of mobility,the emissions from the free fluorescein released by rupture of the cellsor created by hydrolysis in the filtrate are less polarized than that ofbound fluorescein within the lymphocytes. The subtraction of thisartificially high background from the vertically and horizontallypolarized fluorescence intensity measurements on the suspension can leadto the erroneous conclusion that the emissions from thelymphocyte-containing suspension are more polarized than they actuallyare. If the filtration error occurs on a lymphocyte sample that has beenstimulated with a cancer-associated antigen, the result can be a falsenegative test. This occurs because the apparent polarization actuallymeasured is greater than it should be and the decrease in fluorescencepolarization caused by a positive SCM response and indicative of cancerwill be masked. Conversely, if the filtration error occurs on a sampleof unstimulated control lymphocytes, the result can be a false positive.The apparent polarization value of the control is higher than it shouldbe In this case, another sample of lymphocytes from a normal donorexposed to a cancer-associated antigen but not responding to thatantigen gives a lower apparent polarization value if the artificiallyhigh background measurement does not occur on that sample. This lowerapparent polarization value can be interpreted as indicating thepresence of cancer.

There are additional disadvantages associated with the use of thefiltration technique to determine the background for fluorescencepolarization measurements, especially if large-scale clinical testing isintended. Considerable experience and skill are required to extrapolatethe fluorescence measurements accurately and repeatedly over the timeperiod required for filtration. The filtration process is slow,particularly if the pressures used are limited; it requires additionalequipment, and is difficult to carry out reproducibly on more than a fewsamples at a time. Also, the filtration procedure requires rather largevolumes of sample, about 3 ml. Although all of these disadvantages canbe overcome when the SCM test is used for relatively small-scalelaboratory studies, they present serious obstacles to large-scaleclinical use of the SCM test.

Accordingly, there is a need for a fluorescence polarization measurementtechnique capable of compensating for background extracellularfluorescence without using the filtration step. This technique should berapid, suitable for automation, require a small sample, and be capableof being carried out with a minimum of equipment by workers with aminimum of specialized training. Preferably a large number of samplesshould be able to be processed with the technique.

SUMMARY

This invention is directed to methods and apparatus for measuringfluorescence polarization that meet the above needs. The measurementsare conducted on biological structures such as whole cells, particularlylymphocytes, portions of cells such as mitochondria, viruses, orliposomes. The invention is useful whenever the background in which thefluorescing material is dissolved or suspended contributes fluorescencedue to the presence of the same fluor found in the fluorescing material.The invention is particularly useful for measuring fluorescencepolarization on lymphocytes in the SCM test.

The invention is based on the discovery that fluorescence fromintracellular fluorescein in fluorescence-containing lymphocytes isbathochromically shifted (shifted to longer wavelengths) relative to thebackground fluorescence due to extracellular fluorescein. The effect ofthe shift is that the extracellular and intracellular fluorescence canbe regarded as originating from two different fluorophores, with twodifferent fluorescence emission spectra and two different fluorescenceemission maxima. This allows determination of the proportion of thetotal fluorescence intensity at any wavelength attributable to eitherintracellular or extracellular fluorescence.

Most broadly, this invention provides a method for compensating forbackground fluorescence in the measurement of polarized fluorescenceemissions from a fluorescing material in a sample comprising thefluorescing material and background material, the background materialcontributing background fluorescence. The method yields measurementsreflecting the contributions to fluorescence emissions of only thefluorescing material. In the method, there is no need to separatephysically the fluorescing material from the background material.

The fluorescing material is generally isotropic in its response topolarized light since the degree of polarization of the emittedfluorescence relative to that of the exciting light does not depend onthe orientation of the plane-polarized light used to excite thefluorescing material. Our method is therefore applicable whenever theemitted polarized fluorescence is measured in two planes, with thesecond plane being transverse to the first plane. Preferably the firstplane is parallel to the plane of the exciting plane-polarized light.More preferably the two planes are orthogonal. However, because theconventional fluorescence polarization measuring apparatus excites thefluorescing material with vertically polarized light and measures theemitted polarized light in the vertical and horizontal planes, theequations herein describe that orientation of the exciting light and thetwo planes of measurement.

Most generally, the method comprises five basic steps. Step (1)comprises exciting the sample with plane-polarized light. Step (2)comprises measuring the polarized emissions from the sample at a firstor primary wavelength (λ₁). The measurements at λ₁ are made in twoplanes, a first plane parallel to the plane of polarization of theexciting light and a second plane transverse to the first plane. Thesemeasurements yield:

(a) I_(P1), which is the measured fluorescence intensity in the firstplane at λ₁ ; and

(b) I_(T1), which is the measured fluorescence intensity in the secondplane at λ₁.

Step (3) of the method comprises determining at a secondary wavelength(λ₂) different from λ₁ and within the range of wavelengths determined bythe shift of the fluorescence emission spectrum due to backgroundfluorescence, the total intensity of the fluorescence emissions(I_(tot2)).

Step (4) comprises determining from I_(P1), I_(T1), and I_(tot2) thepolarized fluorescence emission intensities in the first plane (I_(P1B))and in the second plane (I_(T1B)) emitted by the background material atλ₁.

Step (5) comprises subtracting I_(P1B) from I_(P1) to obtain I_(P1F) andsubtracting I_(T1B) from I_(T1) to obtain I_(T1F). I_(P1F) and I_(T1F)are the emission intensities in the two planes due solely to thefluorescing material.

Three alternatives exist for the determination of I_(tot2) in thisgeneral method. In the first alternative, measurements of polarizedfluorescence emission intensity are made at λ₂ in the two planes,yielding:

(i) I_(P2), which is the measured fluorescence intensity in the firstplane at λ₂ ; and

1 (i) I_(T2), which is the measured fluorescence intensity in the secondplane at λ₂. In this alternative, I_(P1B) and I_(T1B) are determinedfrom I_(P1), I_(P2), I_(T1), and I_(T2), and I_(tot2) is obtained aspart of the determination of I_(P1B) and I_(T1B).

In the second alternative, I_(tot2), the total intensity of thefluorescence emissions at λ₂, can be measured directly and used todetermine the polarized fluorescence intensities in the first and secondplanes emitted by the background material.

In either the first or second alternatives, the first and second planescan be orthogonal. If so, typically the exciting plane-polarized lightis vertically polarized and the horizontally and vertically polarizedemissions from the sample are measured.

In the third alternative, when vertically polarized light is used toexcite the sample and measurements of the polarized fluorescenceemissions are measured at λ₁ in the vertical and horizontal planes,I_(M2), the polarized fluorescence emissions at λ₂ in a plane oriented54.7° from the vertical, can be measured, whereby the value of I_(M2) isproportional to I_(tot2) regardless of the degree of polarization of thefluorescence emitted by the sample

Preferably λ₁ is chosen to be at the maximum of the fluorescenceemission spectrum of the fluorescing material. λ₂ is then preferablyselected such that the absolute value of ((I_(tot1F)/I_(tot1B))-(I_(tot2F) /I_(tot2B))) is maximized, where:

(a) I_(tot1F) is the total fluorescence emission intensity from thefluorescing material at λ₁ ;

(b I_(tot2F) is the total fluorescence emission intensity from thefluorescing material at λ₂ ;

(c) I_(tot1B) is the total fluorescence emission intensity from thebackground material at λ₁ ; and

(d) I_(tot2B) is the total fluorescence emission intensity from thebackground material at λ₂. This is equivalent to selecting λ₂ such thatthe difference between K_(a) and K_(b) is maximized, where:

(1) K_(a) is a ratio obtained by dividing I_(tot2B) by I_(tot1B), where:

(a) I_(tot2B) is the total fluorescence emission intensity for thebackground material at λ₂ ; and

(b) I_(tot1B) is the total fluorescence emission intensity for thebackground material at λ₁ ;

(2) K_(b) is a similar ratio obtained by dividing I_(tot2F) byI_(tot1F), where:

(a) I_(tot2F) is the total fluorescence emission intensity for thefluorescing material at λ₂ ; and

(b) I_(tot1F) is the total fluorescence emission intensity for thefluorescing material at λ₁. Determination of K_(a) and K_(b) requiresphysical separation of the fluorescing material from the backgroundmaterial. K_(a) and K_(b) are constants for the particularinstrumentation used, but vary with λ₁ and λ₂.

Preferably, the difference between λ₁ and λ₂ is at least about 5 nm andis no greater than about 10 nm and more preferably, about 15 nm. Onlyone secondary wavelength, λ₂, need be used, although more can be used.

When the sample is excited with vertically polarized light and thevertically and horizontally polarized fluorescence emissions therefromare measured, the method can further comprise the step (5) ofdetermining the net polarization value, P, of the fluorescing materialfrom I_(P1F) and I_(T1F) according to the equation:

    P=(I.sub.P1F -GXI.sub.T1F)/(I.sub.P1F +G×I.sub.T1F).

In this equation, G is a correction factor for the unequal transmissionof the vertically and the horizontally polarized fluorescence emissionsthrough the optical system of a fluorescence measuring instrument.

When the exciting light is vertically polarized and the vertically andhorizontally polarized fluorescence emissions are measured, therelationships among the intensities can be described by severalequations. Step (3) can comprise a series of substeps, namely;

(3a): Obtaining the total fluorescence emissions of the sample at λ₁ andλ₂, I_(tot1) and I_(tot2), from I_(P1), I_(T1), I_(P2), and I_(T2) inaccordance with the following relationship:

    I.sub.totλ =I.sub.Pλ +2(I.sub.Tλ ×G).

In this equation:

(i) I_(tot)λ is the total intensity of the fluorescence emissions fromthe sample at the wavelength λ, with λ being equal to either λ₁ or λ₂ ;

(ii) I_(P)λ and I_(T)λ are either I_(P1) and I_(T1) or I_(P2) andI_(T2), depending upon the value of λ; and

(iii) G is a correction factor for the unequal transmission of thevertically and horizontally polarized fluorescence emissions through theoptical system of a fluorescence measuring instrument, and is constantfor any particular fluorescence measuring instrument. As measured, bothI_(P1) and I_(T1) include contributions of fluorescence from both thefluorescing material and the background material.

(3b): Determining a factor F representing the fraction of the totalintensity of the fluorescence emissions at λ₁ due to backgroundfluorescence by the relationship; ##EQU1##

In this equation, K_(a) and K_(b) are constants for the instrumentationused and are determined as stated above, and Q is the ratio of I_(tot2)divided by I_(tot1). Q varies from sample to sample with the relativecontributions to fluorescence of the fluorescing material and thebackground material.

(3c) Determining from the factor F the values of I_(P1B) and I_(T1B)according to the relationships: ##EQU2## wherein P_(k) is a constantdefined by the relationship: ##EQU3##

In the equation defining P_(k), I_(P1BS) and P_(T1BS) are the verticallyand horizontally polarized fluorescence emission intensities,respectively, at λ₁ of the background material in a separate sample fromwhich the fluorescing material has been removed G is the correctionfactor introduced hereinabove in substep (3a).

Additionally and for greater accuracy, this method can make use of atleast one additional secondary wavelength, λ₃, within the range ofwavelengths determined by the shift of the fluorescence emissionspectrum due to background fluorescence emissions. When an additionalsecondary wavelength, λ₃, is used, the method comprises an additionalfour steps.

Of these additional four steps, step (a) is the determination of anumber of quantities at λ₃. These quantities are:

(i) the vertically and horizontally polarized fluorescence emissionsfrom the sample, I_(P3) and I_(T3) ;

(ii) the total fluorescence emission intensity from the backgroundmaterial, I_(tot3B), the fluorescing material having been removed fromthe sample; and

(iii) the total fluorescence emission intensity from the fluorescingmaterial, I_(tot3P).

Step (b) is the determination from I_(P3), I_(T3), I_(tot3B), I_(tot3F),and from the previously determined quantities I_(P1), I_(T1), I_(tot1B),and I_(tot1F), a second value of the factor F, F₂. Step (c) is thedetermination of the average value, F_(av), of the factor F, from thefirst value of F, F₁, which had been previously determined at λ₁ and λ₂and from F₂. Step (d) is the use of F_(av) in determining I_(P1B) andI_(T1B).

The bathochromic shift method of the present invention can be applied tofluor-containing lymphocytes and incorporated directly into the SCM testfor diagnosing the presence of a condition or disease in a body.

The following preparatory steps are needed to incorporate the generalbathochromic shift method into the SCM test:

(1) drawing a sample of lymphocyte-containing body fluid from the bodyto be tested;

(2) separating SCM-responding lymphocytes from the body fluid;

(3) contacting the SCM-responding lymphocytes with an antigen derivedfrom or associated with the disease or condition being tested for tostimulate the lymphocytes;

(4) forming a suspension of the stimulated lymphocytes and a fluorogenicagent precursor; and

(5) maintaining the stimulated lymphocytes in the suspension forsufficient time to allow penetration or the lymphocytes by thefluorogenic agent precursor and the precursor's intracellular hydrolysisto a fluorogenic compound for generating stimulated fluor-containinglymphocytes.

The suspension of the stimulated fluor-containing lymphocytes is thenused as the sample in the general bathochromic shift method, or in themodifications previously described in which I_(tot2) or I_(M2) aremeasured, to obtain I_(P1F) and I_(T1F). These quantities, I_(P1F) andI_(T1F), are then used to determine the SCM response of thefluor-containing lymphocytes to the antigen used to stimulate thelymphocytes. The SCM response of the lymphocytes is an indication of thepresence or absence of the tested for disease or bodily condition in thebody of the donor of the lymphocytes.

The lymphocyte-containing body fluid is typically blood.

The fluorogenic agent precursor is preferably fluorescein diacetate(FDA).

Typically, in the determination of K_(a) and K_(b), physical separationof the fluor-containing lymphocytes from the background material isaccomplished by filtration.

Preferably, when exciting the lymphocytes at 470 nm, λ₁ is 510 nm and λ₂is selected to be between 515 nm and 520 nm or 525 nm, typically 515 nmor 525 nm. Alternatively, when exciting the lymphocytes at 442 nm, λ₁can be 527 nm and λ₂ selected to be between 532 nm and 537 nm or 542 nm,typically 532 nm.

An apparatus useful for practicing the method of this inventioncomprises:

(1) an excitation source for exciting the sample at a selectedexcitation wavelength;

(2) a fixed polarizer transmitting to the sample only plane-polarizedlight, the polarizer being disposed between the light source and thesample;

(3) orientation selection means for selectively transmitting planepolarized light in either a first plane parallel to the plane ofpolarization of the exciting light or a second plane transverse to thefirst plane, the orientation selection means being disposed in the lightpath of the fluorescence emitted by the sample;

(4) wavelength selection means for selecting either the primarywavelength, λ₁ or the secondary wavelength, λ₂, or subsequentfluorescence emission intensity measurements, the wavelength selectionmeans being disposed in the path of the light emitted from thewavelength selection means;

(5) measuring means for measuring the intensities of the components ofthe emitted fluorescence polarized in the first and second planes at thewavelength selected by the wavelength selection means; and

(6) calculation means for calculating the net polarization value, P, ofthe fluorescing material in the sample from the measured intensities.

The excitation means can comprise: (1) a light source; and (2)transmission means for transmitting light at a selected excitationwavelength, the transmission means being disposed in the path of thelight emitted by the light source.

The fixed polarizer can be arranged to transmit only verticallypolarized light to the sample The first and second planes in which theorientation selection means transmits polarized light can then be thevertical and horizontal planes.

The orientation selection means can comprise: (1) a rotatable analyzerdisposed in the light path of the fluorescence emitted by the sample;and (2) means for rotating the polarization axis of the analyzer betweenthe first and second planes.

Alternatively, the orientation selection means can comprise: (1) twoseparate analyzers, a first analyzer with its polarization axis in thefirst plane and a second analyzer with its polarization axis in thesecond plane; and (2) analyzer selection means for alternativelypositioning the first or the second analyzer in the path of thefluorescence emitted by the sample.

The wavelength selection means can be a single device designed to passfluorescence emissions alternately at λ₁ and λ₂. Preferably, however,the wavelength selection means comprises a pair of emissionmonochromators or optical filters, one for each wavelength.

When the wavelength selection means comprises a pair of emissionmonochromators, the apparatus can further comprise:

(1) a pair of photodetectors, one disposed in the exit light path ofeach of the emission monochromators; and (2) an amplifier to which theoutputs from the photodetectors are fed individually. The amplifierincludes suitable conventional switching means to select controllablyfirst one and then the other of the photodetector outputs, whereby theselected output is amplified. In this version of the apparatus, thecalculation means includes a microprocessor to which the selected outputof the amplifier is fed. The microprocessor is programmed to perform thenecessary computations to correct for background fluorescence.Preferably, the microprocessor further includes conversion means toconvert the output of the amplifier to an equivalent digitalrepresentation.

Alternatively, the measuring means can comprise either four separatephotodetectors so that a separate photodetector is dedicated to eachcomponent of the polarized emitted fluorescence at each wavelength, or asingle photodetector.

An alternative version of this apparatus features a rotating polarizerand two fixed analyzers. This version of the apparatus comprises:

(1) an excitation source;

(2) a polarizer disposed between the excitation source and the sample;

(3) a first fixed orientation selection means for selectivelytransmitting plane-polarized light only in a first plane, the firstfixed orientation selection means being disposed in the light path ofthe fluorescence emitted by the sample;

(4) a second fixed orientation selection means for selectivelytransmitting plane-polarized light only in a second plane transverse tothe first plane, the second fixed orientation selection means beingdisposed in the light path of the fluorescence emitted by the sample;

(5) a first wavelength selection means for selecting only a primarywavelength, λ₁, for subsequent fluorescence emission intensitymeasurements, the first wavelength selection means being disposed in thepath of the light emitted from the first fixed orientation selectionmeans;

(6) a second wavelength selection means for selecting only a secondarywavelength, λ₂, for subsequent fluorescence intensity measurements, thesecond wavelength selection means being disposed in the path of thelight emitted from the second fixed orientation selection means;

(7) measuring means for measuring the intensities of the components ofthe emitted fluorescence polarized in the first and second planes at thewavelengths selected by the first and second wavelength selection means;and

(8) calculation means for calculating the net polarization value, P, ofthe fluorescing material in the sample from the measured intensities.

The axis of the rotating polarizer can rotate circularly through anangle of 360° to transmit planepolarized light in all possibleorientations to the sample. In this embodiment, the ratio of thepolarized fluorescence emission intensity measured in the first plane atλ₁ to the polarized fluorescence emission intensity measured in thesecond plane at λ₂ varies sinusoidally with the rotation of thepolarizer through the angle of 360°. Alternatively, the axis of therotating polarizer can rotate through an angle of 90° and fluorescenceintensity measurements can be taken at the extremes of rotation of thepolarizer, such that the second plane is orthogonal to the first plane.

Another alternative version of this apparatus measures I_(tot2)directly, eliminating the need for separate measurements of I_(P2) andI_(T2). This alternative version comprises:

(1) an excitation source;

(2) a fixed polarizer;

(3) orientation selection means for selectively transmitting planepolarized light in either a first plane or the second plane, theorientation selection means being movably disposed alternately in thelight path of the fluorescence emitted by the sample or outside of thelight path;

(4) wavelength selection means;

(5) positioning means interlocked with the wavelength selection meanssuch that the positioning means positions the orientation selectionmeans in the light path only whenever the wavelength selection meansselects λ₁ and positions the orientation selection means outside thelight path whenever the wavelength selection means selects λ₂ ;

(6) a first measuring means for measuring the intensity of thecomponents of the emitted fluorescence polarized in the first and secondplanes at λ₁ whenever the orientation selection means is disposed in thelight path;

(7) a second measuring means for measuring I_(tot2), the total intensityof the fluorescence emitted from the sample at λ₂ whenever theorientation selection means is outside of the light path; and

(8) calculation means.

The orientation selection means can comprise: (1) a rotatable analyzerdisposed in the light path of the fluorescence emitted by the samplewhen the orientation selection means is disposed in the light path; and(2) rotation means.

Alternatively, the orientation selection means can comprise (1) twoseparate analyzers; and (2) analyzer selection means for alternatelypositioning the first analyzer or the second analyzer in the light pathof the fluorescence emitted by the sample when the orientation selectionmeans is positioned in the light path.

The wavelength selection means can be a single device or a pair ofdevices, in either case interlocked with the positioning means. Thewavelength selection means can be emission monochromators.

When the wavelength selection means are emission monochromators, thefirst measuring means can comprise a first photodetector disposed in theexit light path of the first emission monochromator and measuring thecomponent of the fluorescence transmitted by the orientation selectionmeans, thereby alternately measuring the component of the fluorescenceemissions polarized in the first and second plane at λ₁. The secondmeasuring means comprises a second photodetector disposed in the exitlight path of the second monochromator, and measuring the totalintensity of the fluorescence emissions at λ₂. This embodiment of theapparatus also includes an amplifier as described above, with thecalculation means similarly including a microprocessor Themicroprocessor preferably further includes conversion means to convertthe output of the amplifier to an equivalent digital representation.

Another version of the apparatus is similar but includes threephotodetectors. In this version, the first measuring means comprises twoseparate photodetectors:

(1) a first photodetector disposed in the exit light path of the firstemission monochromator to which the light transmitted by the orientationselection means when the orientation selection means is in the lightpath and transmits plane polarized light in the first plane is directed;and

(2) a second photodetector disposed in the exit light path of the firstemission monochromator to which the light transmitted by the orientationselection means when the orientation selection means is in the lightpath and transmits plane polarized light in the second plane isdirected. The first photodetector measures the component of thefluorescence emissions polarized in the first plane at λ₁, while thesecond photodetector measures the component of the fluorescenceemissions polarized in the second plane at λ₁. In this arrangement, asbefore, the second measuring means comprises a separate photodetector,here the third photodetector.

Still another basic version of this apparatus makes use of the findingthat when the orientation of the polarization axis of the analyzer is atan angle of 54.7° from the plane of polarization of the exciting light,the polarized fluorescence emission intensity measured in this plane ata given wavelength is proportional to the total fluorescence emissionintensity at that wavelength, regardless of the degree of polarizationof the fluorescence emitted by the sample. When this measurement is madeat λ₂, the measured polarized fluorescence intensity is designatedI_(M2) the total fluorescence emission intensity determined therefrom isI_(tot2).

This version of the apparatus comprises:

(1) an excitation source;

(2) a fixed polarizer;

(3) orientation selection means for selectively transmitting planepolarized light in one of three planes: a vertical plane, a horizontalplane, and a plane oriented 54.7° from the vertical, the orientationselection means disposed in the path of the fluorescence emitted by thesample;

(4) wavelength selection means for selecting either λ₁ or λ₂ forsubsequent fluorescence emission intensity measurements, the wavelengthselection means interlocked with the orientation selection means so thatλ₁ is selected whenever the orientation selection means transmits lightin either the vertical plane or the horizontal plane, and so that λ₂ isselected whenever the orientation selection means transmits light in theplane oriented 54.7° from the vertical;

(5)measuring means for measuring the intensities of the components ofthe emitted fluorescence polarized in the vertical plane, the horizontalplane, and the plane oriented 54.7° from the vertical, at the wavelengthselected by the wavelength selection means; and

(6) calculation means for determining I_(tot2) from I_(M2) andcalculating P.

The orientation selection means can comprise: (1) a rotatable analyzer;and (2) means for rotating the orientation of the polarization axis ofthe analyzer between the first, second, and third planes.

Alternatively, the orientation selection means can comprise: (1) threeseparate analyzers, each with its polarization axis in a different oneof the three planes; and (2) analyzer selection means for alternatelypositioning the first, second, or third analyzer in the path of thefluorescence emitted by the sample.

In this version of the apparatus, the measuring means can comprise asingle photodetector, two photodetectors, or three photodetectors. Whenthe wavelength selection means is a pair of emission monochromators, themeasuring means can comprise either two or three photodetectors inslightly different arrangements.

When two photodetectors are used, the first photodetector can bedisposed in the exit light path of the first emission monochromator andmeasure alternately the component of the fluorescence transmitted by theorientation selection means in the vertical and the horizontal planes atλ₁. The second photodetector is disposed in the exit light path of thesecond emission monochromator and measures the component of thefluorescence transmitted by the orientation selection means whenever thepolarization axis of the analyzer is in the plane oriented 54.7° fromthe vertical.

When three separate photodetectors are used, a single photodetector isdedicated to the measurement of the polarized fluorescence emissions ineach of the three planes.

Whether two or three photodetectors are used, the apparatus also furthercomprises an amplifier as described above, with the calculation meanssimilarly including a microprocessor.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription, appended claims, and accompanying drawings where:

FIG. 1 is a flow chart of a method in accordance with the presentinvention useful in the general SCM test procedure;

FIG. 2 is a flow chart of the general SCM test procedure as applied inthe method of the present invention in FIG. 1;

FIG. 3 is a schematic diagram of a fluorescence spectrophotometerarrangement utilized for measurement of the SCM response of a lymphocytesuspension;

FIG. 4 is a schematic diagram of an alternative fluorescencespectrophotometer arrangement for measurement of the SCM response inwhich the polarizer rotates and two fixed analyzers are used to transmitthe polarized fluorescence emissions from the sample in the first andsecond planes;

FIG. 5 is a schematic diagram of an alternative fluorescencespectrophotometer arrangement utilized for measurement of the SCMresponse in which the total fluorescence emission intensity at λ₂,I_(tot2), is measured directly; and

FIG. 6 is a schematic diagram of another alternative fluorescencespectrophotometer arrangement in which the polarized fluorescenceemission intensity is also measured at λ₂ in a plane at an angle 54.7°from the plane of the exciting light, allowing direct calculation ofI_(tot2).

DESCRIPTION

This invention relates to methods for correcting fluorescencepolarization measurements of suspensions of fluor-containing cells suchas lymphocytes for extracellular background fluorescence withoutfiltering the cells from the suspension. The methods are based on theobservation that the fluorescence from the fluor-containing cells isbathochromically shifted (shifted to longer wavelengths) relative to thefluorescence from the extracellular background and, therefore theextracellular and the intracellular fluorescence can be regarded asoriginating from two different fluorophores. The method is notrestricted to the analysis of fluorescence emissions fromfluor-containing cells in a background of extracellular fluorescence; itis equally applicable to any fluorescent system in which thefluorescence from an environment contributing background fluorescence isshifted in wavelength with respect to the fluorescence from thefluorescing material.

One potential application of the bathochromic shift method of thepresent invention is in flow cytometry and microscopic observation ofsingle cells. When these techniques are used with fluorescencepolarization, a halo is often observed around cells because ofconcentration gradients in the medium near the cells. This halo effectis enhanced when the permeability of the cell membrane is increased, aswhen lymphocytes are stimulated by mitogens such as PHA. The method ofthe present invention can eliminate this halo effect by correcting forthe background fluorescence giving rise to it.

Theoretical Background

Fluorescence from intracellular molecules of fluorescein isbathochromically shifted (shifted to longer wavelengths) by about 9 nmrelative to the fluorescence from fluorescein in aqueous phosphatebuffered saline. The extracellular and intracellular fluoresceinfluorescence, therefore, can theoretically be treated as originatingfrom two different fluorophores. On this basis the followingmathematical analysis can be applied:

    I.sub.tot1 =I.sub.tot1B +I.sub.tot1F                       (1)

    I.sub.tot2 =I.sub.tot2B +I.sub.tot2F                       (2)

where:

(a) I_(tot1) is the total intensity of fluorescence at the wavelength λ₁;

(b) I_(tot2) is the total intensity of fluorescence at the wavelength λ₂;

(c) I_(tot1B) is the total intensity of fluorescence at the wavelengthλ₁ ;

(d) I_(tot2B) is the total intensity of fluorescence from theextracellular fluorescein at the wavelength λ₂ ;

(e) I_(tot1F) is the total intensity of fluorescence from theintracellular fluorescein at the wavelength λ₁ ; and

(f) I_(tot2F) is the total intensity of fluorescence from theintracellular fluorescein at the wavelength λ₂. The term "total" refersto the fluorescence intensity, whether polarized in a particular planeor not. λ₁ and λ₂ are two different wavelengths, for example 510 nm and515 nm respectively. λ₁ is designated as the first or primarywavelength; all other wavelengths at which measurements are taken,including λ₂, are designated secondary wavelengths.

In general, the fluor-containing cells subjected to fluorescencepolarization are isotropic in their response to polarized light. Thatis, the degree of polarization of the emitted fluorescence relative tothat of the exciting light does not depend on the orientation of theplane-polarized light used to excite the cells. This isotropic responsemakes the methods described herein applicable whenever the emittedpolarized fluorescence is measured in two planes, a first plane and asecond plane transverse to the first plane. The fluorescencepolarization intensities in these two planes are designated I_(P) in thefirst plane and I_(T) in the second plane. Typically, the first plane isparallel to the plane of polarization of the exciting light, but this isnot a requirement for the application of the method. Preferably the twoplanes are orthogonal.

The fluorescence polarization measuring apparatus used for the methodsof this invention uses vertically polarized light to excite thefluor-containing cells and measures the emitted polarized light in thevertical and horizontal planes. Therefore the equations used to definethe process of determining fluorescence polarization from thesemeasurements are written in terms of vertically polarized excitinglight, with measurements of the emitted polarized light being made invertical and horizontal planes. In these equations, the vertical planeis the first plane, and the horizontal plane is the second or transverseplane. For simplicity, the intensities in the vertical and horizontalplanes will continue to be designated I_(P) and I_(T) in the followingequations, as this relationship is merely a special case of the moregeneral parallel-transverse relationship of the two planes.

When the SCM test is performed, the total intensity of the fluorescenceat a particular wavelength need not necessarily be measured. Rather whatcan be measured are the intensities of the vertically and thehorizontally polarized components of the fluorescence at a particularwavelength. The total fluorescence intensity at a particular wavelengthis determined by the equation

    I.sub.tot1 =I.sub.Pλ +2(I.sub.Tλ ×G),  (3)

as previously described in the Summary. Alternatively, only theintensities of the vertically and the horizontally polarized componentsof the fluorescence at the primary wavelength can be measured, and theapparatus can be arranged to measure the total intensity of thefluorescence at the secondary wavelength. This is feasible because onlythe total intensity of the fluorescence at the secondary wavelength isactually needed for the performance of the method.

If the following quantities are defined in terms of previously definedquantities:

    K.sub.a =I.sub.tot2B /I.sub.tot1B ;                        (4)

    K.sub.b =I.sub.tot2F /I.sub.tot1F ; and                    (5)

    Q=I.sub.tot2 /I.sub.tot1,                                  (6)

then the fraction, F, of the total intensity of the fluorescenceemissions at the primary wavelength, λ₁, due to extracellular emissionsis given by: ##EQU4##

Once F is determined the vertically and horizontally polarized emissionintensities at the primary wavelength due to extracellular fluorescencecan be determined by the equations: ##EQU5## where: (a) I_(P1B) is thevertically polarized extracellular fluorescence intensity at the primarywavelength λ₁ ;

(b) I_(T1B) is the horizontally polarized extracellular fluorescenceintensity at the primary wavelength;

(c) I_(tot1) is the total intensity of the fluorescence emissions at theprimary wavelength as calculated from equation (3);

(d) F is the fraction of the total intensity of the fluorescenceemissions at the primary wavelength due to extracellular fluorescence ascalculated from equation (7); and

(e) P_(k) is a constant defined by the relationship: ##EQU6## where: (i)I_(P1BS) is the vertically polarized fluorescence intensity at theprimary wavelength of a separate solution of a nonfluorogenic compound,such as FDA, hydrolyzable intracellularly to a fluorogenic compound,such as fluorescein, from which the lymphocytes have been removed,preferably by filtration:

(ii) I_(T1BS) is the horizontally polarized fluorescence intensity atthe primary wavelength of such a separate solution; and

(iii) G is the correction factor for the unequal transmission of thevertically and horizontally polarized components of the fluorescenceemissions through the optical system of the fluorescence measuringinstrument from equation (3). P_(K) is equivalent to the extracellularfluorescein fluorescence polarization and is a constant equal to 0.0254at 27° C.

Once I_(P1B) and I_(T1B) are known, the vertically and horizontallypolarized emission intensities at the primary wavelength due solely tointracellular fluorescence, I_(P1F) and I_(T1F), can be readilycalculated as:

    I.sub.P1F =I.sub.P1 -I.sub.P1B                             (11)

    I.sub.T1B =I.sub.T1 -I.sub.T1B,                            (12)

where I_(P1) and I_(T1) are the polarized fluorescence intensitiesmeasured in the first and second planes, respectively, at λ₁.

Once I_(P1F) and I_(T1F) are known, the net polarization value, P, canbe calculated as:

    P=(I.sub.P1F -GXI.sub.T1F)/(I.sub.P1F +GxI.sub.T1F).       (13)

It is the P value that is relevant for the SCM test and whose decreasein response to the challenge of the lymphocytes by an antigen associatedwith a disease or condition signals a positive response to the SCM test.

It is necessary to determine the constants K_(a), K_(b), and P_(k), aswell as G. The first three of these are constants for a particular celltype fluorogenic molecule, and fluorescence measuring instrument. G isconstant for a particular fluorescence measuring instrument regardlessof the cell type or fluorogenic molecule used. K_(a), K_(b), and P_(k)are all determined by performing the prior art kinetic extrapolation andfiltration method on a sample at at least two different wavelengths, oneprimary wavelength and at least one secondary wavelength, so that theextracellular and intracellular fluorescence intensities at thesewavelengths can be determined. G, if not known from previousfluorescence measurements on the same instrument, can be determined frommeasurements of the vertically and horizontally polarized fluorescenceintensities of the filtrate or a solution of the fluorogenic moleculeexcited with horizontally polarized light.

The process of using the method of the present invention is summarizedin the flowchart of FIG. 1 Step 1 is to determine at least one secondarywavelength. Step 2 is to determine the instrument constants K_(a),K_(b), and P_(k), as well as G if not previously determined. Step 3 isto measure the vertically and horizontally polarized fluorescenceemission intensities at these same wavelengths--the primary wavelengthand at least one secondary wavelength. In step 4, these intensitymeasurements are then used to determine I_(tota1) for each wavelengthaccording to equation (3) for each sample. These values of I_(tota1) atthese wavelengths are then used in step 5 to determine a value of Q foreach sample according to equation (6). The value of Q varies for eachsample according to the relative contributions of intracellular andextracellular fluorescence in that sample, because the intracellular andextracellular fluorescence emissions are regarded as having differentspectra. Therefore, the actual fluorescence emission spectrum observedcan be regarded as the algebraic superposition of the two spectra,weighted for the relative contributions of each. Q is the only factorvarying from sample to sample in a series of measurements on the samecell type using the same intracellular fluorogenic molecule and measuredin the same fluorescence measurement apparatus.

Once a value of Q is obtained for each sample, in step 6 this value isused in equation (7) along with K_(a) and K_(b) to calculate a value ofF for each sample. This value of F is then used in step 7 to determineI_(P1B) and I_(T1B). From these values the vertically and horizontallypolarized fluorescence emission intensities at the primary wavelengthdue to intracellular fluorescence, I_(P1F) and I_(T1F) are thendetermined in step 8 for each sample In the last step, step 9, theselatter values can then be used directly in equation (13) to calculatethe net polarization value P for each cell sample.

2. Use of the Bathochromic Shift Method in Performing the SCM Test

The general procedure of the SCM test is summarized in the flowchart ofFIG. 2.

a. Isolation of Potentially SCM-Responding Lymphocytes

To perform the SCM test, the subpopulation of lymphocytes described as"potentially SCM-responding lymphocytes" is separated from theperipheral blood to be tested. This separation can be performed by themethods described in L. Cercek and B. Cercek, "Application of thePhenomenon of Changes in the Structuredness of Cytoplasmic Matrix (SCM)in the Diagnosis of Malignant Disorders: a Review," Europ. J. Cancer 13903-915 (1977), and in the prior patent application by the Cerceksentitled "Automated Collection of Buoyant Density Specific Cells fromDensity Gradients," Ser. No. 838,264, filed Mar. 10, 1986, andincorporated herein by this reference. These methods, with reference toFIG. 2, basically involve a first step of removing the phagocytic cellsby treating the lymphocytes with iron powder or carbonyl-iron powder andthen placing the cells on a magnet to effect separation of thephagocytic cells along with the iron powder from the blood sample. Theiron powder separation step can be replaced by other suitable methods ofeliminating the phagocytic cells from the blood sample. After thephagocytic cells are removed, the potentially SCM-responding lymphocytesare isolated by centrifuging the lymphocytes through a Ficoll.sub.(TM)-Triosil.sub.(TM) or Percoll.sub.(TM) density gradient solution. Thelymphocytes characterized as "potentially SCM-responding lymphocytes"have a buoyant density of about 1.059 g/cm³ to about 1.067 g/cm³ at 20°C. at an osmolality of about 0 315 to about 0.320 Osm/kg. Afterseparation and washing a portion of the potentially SCM-respondinglymphocytes is retained as a control and other portions are subsequentlystimulated by incubation with a challenging agent such as an antigenderived from or associated with the disease or condition being testedfor.

b. General Procedures for Performing the SCM Test

Once the appropriate lymphocytes have been isolated, the SCM test isperformed according to the methods described in the European Journal ofCancer article, supra. The SCM test measures the decrease influorescence polarization after the lymphocytes have been incubated witha challenging agent such as a cancer-associated antigen or an antigenassociated with another disease or condition being tested for, or,alternatively, with a mitogen such as phytohaemagglutinin, concanavalinA, or pokeweed mitogen. When the disease to be tested for is cancer,lymphocytes from cancer patients respond with a decrease in the measuredfluorescence polarization to cancer-associated antigens and not tomitogens, while lymphocytes from persons free of malignant diseaserespond only to mitogens and not to the cancer-associated antigens. Tocompare the response of the lymphocytes to the antigen and to themitogen, an SCM response ratio, RR_(SCM), can be determined. TheRR_(SCM) is defined as:

    RR.sub.SCM =P.sub.λ /P.sub.M,                       (14)

where P.sub.λ is the net polarization value for the lymphocytes afterstimulation with the antigen associated with the disease to be testedfor, such as a cancer-associated antigen, and P_(M) is the netpolarization value for another aliquot of the same lymphocytes afterstimulation with a mitogen, preferably phytohaemagglutinin PHA).

To ensure the reproducibility of results, using FDA as the fluorogenicagent precursor, the pH of the FDA substrate solution is maintained ator slightly above 7.4 and the osmolality of the solution should be heldto within 1% of the isotonic value of 0 330 Osm/kg. The selection ofexcitation and emission wavelengths is a matter of choice; the optimalwavelength depends on the fluorogenic agent employed. When FDA is usedas the fluorogenic agent precursor, good results have been obtainedusing an excitation wavelength of 470 nm and an emission wavelength of510 nm. Good results have also been achieved using an excitationwavelength of 442 nm and an emission wavelength of 527 nm using FDA asthe fluorogenic agent precursor. Other fluorogenic agent precursorsusable include dichlorofluorescein diacetate and trichlorofluoresceindiacetate. The fluorogenic agent precursor carboxyfluorescein diacetatedoes not enter mitochondria, but can be useful for observation of eventsaffecting the SCM and occurring in the cytoplasm or other organelles.

These measurements of the SCM on cell suspensions are conducted in afluorescence polarization measuring apparatus comprising a fluorescencespectrophotometer equipped with a polarizer designed to pass verticallypolarized light between the excitation monochromator or optical filterand the cell sample. The apparatus typically includes a rotatableanalyzer capable of passing either vertically or horizontally polarizedlight between the cell sample and the emission monochromator. Althoughthe spectrophotometer can be equipped with a single photodetector suchas a photomultipIier and with means for sequentially measuring theemissions at two different wavelengths, it is preferred that thespectrophotometer be equipped with at least two separatephotomultipliers so that emissions at two wavelengths can be measuredsimultaneously.

3. Apparatus for the SCM Method

Referring to FIG. 3 there is a schematic illustration of one version ofan apparatus useful for measuring SCM on cell suspensions in accordancewith the method of the invention. The excitation source 10 can comprisea suitable light source such as a xenon lamp as well as a transmissiondevice such as an optical filter or, as shown, an excitationmonochromator 12, disposed in the excitation light path of the lightsource for transmitting light to the sample at a selected excitationwavelength. Alternatively, the excitation source itself can be a sourcesuch as a tunable laser adjusted to emit light only at the desiredexcitation wavelength, in which case the separate transmission device isnot needed. In either case, the light is transmitted to a fixedpolarizer 14 that transmits only plane-polarized light to the samplecell suspension 16. The sample cell suspension 16 containsintracellularly the fluorogenic compound such as fluorescein produced byhydrolysis of a nonfluorogenic compound such as FDA. Upon exposure tothe plane-polarized excitation light the fluorescein molecules fluoresceand the fluorescence emission is directed through an orientationselection device. The orientation selection device can comprise, asshown in FIG. 3, an analyzer 18 fitted with a rotator such as anautomatic position changer (not shown) for rotating the orientation ofthe polarization axis of the analyzer 18 between a first plane parallelto the plane of polarization of the exciting light and a second planetransverse to the first plane, whereby the analyzer 18 alternatelytransmits the component of the fluorescence emitted by the samplepolarized in the first plane and the second plane. Alternatively, theorientation selection device can comprise two separate analyzers and ananalyzer selection mechanism for alternately positioning the firstanalyzer or the second analyzer in the path of the fluorescence emittedby the sample 16. In this arrangement, the first analyzer has itspolarization axis in the first plane and the second analyzer has itspolarization axis in the second plane. In either case, the fluorescenceemission, being divided into these two components, then passes throughwavelength selectors such as optical filters, or as illustrated, a pairof emission monochromators 20 and 22, one of which is set to pass onlylight at the first or primary wavelength, λ₁, and the other to passlight at the secondary wavelength, λ₂. A pair of photodetectors 24 and26, which can be photomultiplier tubes, are placed in the exit lightpaths of the emission monochromators 20 and 22 respectively. The outputsfrom the photodetectors 24 and 26 are fed individually to an amplifier28. The amplifier 28 includes suitable conventional switching devices toselect controllably first one and then the other of the photodetectoroutputs. The selected output is amplified and fed to a suitablemicroprocessor 30. Preferably, the microprocessor 30 includes circuitryto convert the analog output of the amplifier 28 to an equivalentdigital representation. The microprocessor 30 is programmed in anotherwise conventional fashion to perform the necessary mathematicalcomputations to correct for extracellular fluorescence as describedabove under "Theoretical Background." The output from the microprocessor30 is then sent to a printer 32 for recording the computed intracellularfluorescein fluorescence polarization values.

An alternative embodiment of the apparatus as shown in FIG. 4 has arotating polarizer 60 and two fixed analyzers 62 and 64 set to passlight in the first and second planes respectively. The polarizer rotatesover 360° to transmit plane-polarized light in all possible orientationsto the sample. In this arrangement, the fluorescence emissions passingthrough the first fixed analyzer 62 then pass through a wavelengthselector such as an optical filter, or, as illustrated, an emissionmonochromator 66 set to pass light only at λ₁. The polarizedfluorescence emissions passing through the second fixed analyzer 64 thenpass through an equivalent wavelength selector such as the emissionmonochromator 68 set to pass light only at λ₂. The arrangement of thephotodetectors 24 and 26, the amplifier 28, the microprocessor 30, andthe printer 32 is the same as in the embodiment shown in FIG. 3. In theembodiment with a rotatable polarizer 60, the polarized fluorescenceintensities measured by the photodetectors 24 and 26 vary sinusoidallywith the rotation of the polarizer 60.

Other alternative embodiments are possible using a rotating polarizerand fixed analyzers. The polarizer need not rotate over a full 360°, butcan rotate over any lesser angle. For example, the polarizer can rotateover 90° with measurements being taken at the extremes of rotation ofthe polarizer. This gives a second plane orthogonal to the first plane.

The fluorescence spectrophotometer utilized has high sensitivity, beingcapable of detecting fluorescence emissions from a concentration of10⁻¹⁰ M fluorescein, and high stability. The spectrophotometer is alsoable to compensate for fluctuations in the intensity of the excitationlight. This compensation ability is important because the intensities ofthe polarized components of the fluorescence are recorded as a functionof time and the bulk concentration of fluorescein in the SCMmeasurements is only of the order of 10⁻⁸ M to 10⁻⁹ M. Also. instrumentsemploying broad band optical filters or monochromators cannot be used inthe SCM measurements since the excitation and emission polarizationspectra of the lymphocytes show that changes in the SCM can be detectedonly within a narrow wavelength region. The spectrophotometer preferablyis also fitted with a thermostatically controlled cuvette holder sincethe fluorescein polarization values are highly temperature dependent,changing by about 3% per degree Celsius change in temperature.Therefore, to ensure the reproducibility of results the temperature ofthe sample should be closely controlled to plus or minus 0.2° C. Thespectrophotometer should have bandwidth and stability characteristicscomparable to the Perkin-Elmer model MPF-4 fluorescencespectrophotometer.

Rather than using the pair of photodetectors 24 and 26 for measurementof the vertical and horizontal fluorescence intensity components at theprimary wavelength and at a secondary wavelength, as many as fourseparate photodetectors can be used. This allows a separatephotodetector to be dedicated to the horizontally and verticallypolarized components of fluorescence at each of the two wavelengthsbeing measured. Alternatively, a single photodetector can be used tomeasure the horizontally and the vertically polarized components offluorescence. Also, a single device such as a filter designed to passfluorescence emissions alternately at the primary wavelength and asecondary wavelength can be employed in place of two filters ormonochromators.

Other versions of this apparatus are described below under"Simplification of the Bathochromic Shift Method." These versions can beemployed when I_(tot2) is measured directly in place of I_(P2) andI_(T2), or when I_(tot2) is determined directly from measurements madein a third plane oriented 54.7° (the so-called "magic angle") from theorientation of the plane of the polarization of the exciting light suchthat the intensity of the polarized fluorescence emissions measured inthis plane at λ₂ (I_(M2)) is proportional to I_(tot2) regardless of thedegree of polarization of the emitted fluorescence.

4. Selection of Wavelengths and Determination of Constants for theBathochromic Shift Method

As outlined in the section entitled "Theoretical Background," thebathochromic shift method of the present invention requires theselection of at least two wavelengths at which the intensity ofhorizontally and vertically polarized emissions are measured, a primarywavelength and at least one secondary wavelength. The primary wavelengthis preferably one of the emission wavelengths in the excitation andemission wavelength combination described above, in the section entitled"General Procedures for Performing the SCM Test." For example, if anexcitation wavelength of 470 nm is selected, the primary wavelength, λ₁,is preferably 510 nm. Similarly, if an excitation wavelength of 442 nmis selected, the primary wavelength is preferably 527 nm. For thepurpose of the following description, the excitation-emission wavelengthcombination of 470 nm and 510 nm is used and the primary wavelength isthus 510 nm.

The secondary wavelength, λ₂, for fluorescence emission measurement isselected to be within a range of wavelengths relatively close to theprimary wavelength, λ₁. The range of wavelengths is determined by theshift of the fluorescence emission spectrum due to backgroundfluorescence emissions. A secondary wavelength within a range of about 5nm to about 15 nm above the primary wavelength has been found to produceacceptable results. Once λ₁ and λ₂ are decided, K_(a) and K_(b) can thenbe calculated. The constant K_(a) is obtained by measuring thehorizontally and vertically polarized fluorescence intensities atprimary and secondary wavelengths of filtrates of SCM-respondinglymphocyte suspensions after incubation in the FDA solution. Foradequate accuracy, these measurements are repeated many times,preferably times or more. For each set of measurements, the totalfluorescence emission intensity is determined at each wavelength bymeans of equation (2) above. The constant K_(a) is then obtained as themean value of the result obtained by dividing the intensity at thesecondary wavelength by the intensity at the primary wavelength for eachset of measurements. Similarly, the constant K_(b) is obtained bymeasuring the horizontally and vertically polarized fluorescenceintensities at primary and secondary wavelengths of the SCM-respondinglymphocytes themselves after correction for the extracellular backgroundfluorescence. The total fluorescence emission intensity at eachwavelength for each set of measurements is then calculated. The constantK_(b) is then obtained as the mean value of the result obtained bydividing the intensity at the secondary wavelength by the intensity atthe primary wavelength for each set of measurements. The constants K_(a)and K.sub. b are recalculated for each different instrument upon whichthe method of the present invention is used and for each new fluorogenicagent used in such method.

The secondary wavelength is preferably selected such that the differencebetween the constants K_(a) and K_(b) is maximized. Such a selectionprocess can include, for example, determining K_(a) and K_(b) for aplurality of wavelengths between 5 nm and 15 nm above the primarywavelength and selecting the secondary wavelength such that thedifference between K_(a) and K_(b) has the largest absolute value.Varying the wavelength by, for example, 5 nm during this selectionprocess e.g., 5 nm, 10 nm, and 15 nm) provides suitable resolution forthe purpose of selecting the difference between the primary andsecondary wavelenqth yielding the largest absolute difference betweenK_(a) and K_(b). As an example, with an excitation wavelength of 470 nmand a primary wavelength of 510 nm, a secondary wavelength of 525 nm wasfound to give the maximum absolute difference between K_(a) and K_(b)and yielded values for K_(a) and K_(b) of 1.0393 and 1.2546respectively. Using the same spectrophotometer, excitation wavelengthand primary wavelength, a secondary wavelength of 515 nm, giving adifference between the primary and secondary wavelength of 5 nm, gave aK_(a) of 1 048 and a K_(b) of 1.135.

A third constant related to the specific instrument used with thepresent invention is the extracellular fluorescence polarization value,P_(k), determined using equation (10). This equation makes use of thevertically and horizontally polarized fluorescence emission intensitiesmeasured at the primary wavelength for the filtrate. G is a correctionfactor for the unequal transmission of the vertically and horizontallypolarized components of the fluorescence through the optical measuringsystem of the fluorescence measuring instrument. The value of G isdetermined by dividing the intensity of the vertically polarizedcomponent of the fluorescence by the intensity of the horizontallypolarized component of the fluorescence emitted from either a filtratesolution or a 10⁻⁷ M solution of fluorescein in phosphate bufferedsaline which has been excited with horizontally polarized light of thesame wavelength as the vertically polarized exciting light. For thePerkin-Elmer MPF-4 fluorescence spectrophotometer utilized herein,G=0.42. This value of G yields a value for P_(k) of 0.0254 at 27° C.

With the constants K_(a), K_(b), and P_(k) determined, the background orextracellular fluorescence at the primary wavelength can be determinedin accordance with the present invention. These constants can bedetermined for a particular fluorogenic agent and particular instrumentand stored in the instrument for use, as in the automated performance ofthe method of the present invention. Once these constants aredetermined, all that the method requires is the measurement of theintensity of the vertically and horizontally polarized components of thefluorescence emissions from the fluor-containing cell sample at theprimary wavelength and at least one secondary wavelength. This thenallows the ready calculation of the P value for the particular cellsample.

5. Extensions of the Bathochromic Shift Method a. Use of More Than OneSecondary Wavelength

Although good results are obtained by the use of the bathochromic shiftmethod when fluorescence measurements are made at only one secondarywavelength, greater accuracy can be achieved when fluorescencemeasurements to compensate for background fluorescence emissions aremade at a plurality of secondary wavelengths distributed through awavelength shift range of about 5 nm to about 15 nm above the primarywavelength. For example, measurements are made not only at the primarywavelength of 510 nm and the first secondary wavelength of 515 nm, butalso at additional secondary wavelengths, for example, 518 nm, 522 nm,and 525 nm, distributed throughout the 5 nm to 15 nm wavelength shiftrange. The additional secondary wavelengths are designated λ₂, λ₄, . . ..

When a plurality of secondary wavelengths is used, the data analysismethod described herein is employed in the same manner as when only onesecondary wavelength is used. Equation (7) is used to calculate thefactor F for each of the individual secondary wavelengths employed foreach sample. The mean value of F for each sample is then determined fromthe values of F for each of the secondary wavelengths used, and the meanvalue of F thus determined is then used in equations (8) and (9) tocalculate I_(P1B) and I_(T1B). Those values are then used in the rest ofthe analysis as previously described to determine the polarizationvalues for the samples.

b. Additional Applications of the Method

The bathochromic shift method is not limited to its application to theSCM test with lymphocytes. It can be used in any biological system inwhich polarized fluorescence is to be measured from cells or otherbiological structures such as organelles, viruses, or liposomes, in thepresence of a background contributing fluorescence. The method asextended has two principal requirements: (1) the fluor used must havedistinguishable spectra when incorporated in or bound to the biologicalstructure and when present in the background, and (2) the constantsK_(a), K_(b), and P_(k) must be capable of being determined. Thedetermination of these constants can involve physical separation of thebiological structure from the background material. This separation canbe performed by filtration, as in the application to the SCM test withlymphocytes. Alternately, the separation can be performed by techniquessuch as centrifugation, gel filtration chromatography, or reversibleprecipitation of the fluor-containing structures. Also, a measurement ofthe vertically and horizontally polarized emission intensities at theprimary wavelength due solely to the background must be made. Thismeasurement can be made on a filtrate, as in the application of thebathochromic shift method to lymphocytes in the SCM test Equally well,this measurement can be made on a supernatant if the fluor-containingstructure is separated out by centrifugation or precipitation.

c. Simplification of the Bathochromic Shift Method

If K_(a), K_(b), P_(k), and G are known, the only use made of the valuesof I_(P2) and I_(T2), the polarized fluorescence emission intensitiesmeasured in the first and second planes at λ₂, for a particular sampleis in the determination of I_(tot2), the total fluorescence intensity atλ₂. I_(tot2) is then used in the determination of Q and therefore F forthat sample. If I_(tot2) is measured or determined directly, there isthen no need to measure I_(P2) and I_(T2) separately and thebathochromic shift method of the present invention can be simplified byeliminating the separate measurements of I_(P2) and I_(T2).

(1) Direct Measurement of I_(tot2)

I_(tot2) can be measured directly and then used to determine Q and thenF using a modification of the fluorescence polarization apparatuspreviously described. An apparatus suitable for the direct measurementof I_(tot2) as well as I_(P1) and I_(T1) is shown in FIG. 5. Thisapparatus has an orientation selection device for selectivelytransmitting plane polarized light in either a first plane parallel tothe plane of polarization of the exciting light or a second planetransverse to the first plane. The orientation selection device ismovably disposed alternately in the light path of the fluorescenceemitted by the cell suspension 16 or outside of the light path. Theorientation selection device can comprise a rotatable analyzer 18 and ananalyzer rotator 44 for rotating the polarization axis of the analyzerbetween the two planes. A wavelength selector 40, shown as the pair ofemission monochromators 20 and 22, alternately selects λ₁ or λ₂. Apositioner 42 interlocks with the wavelength selector 40 such that thepositioner 42 positions the orientation selection device, including theanalyzer 18, within the light path of the fluorescence emitted by thesample only whenever the selector 40 selects λ₁. The positioner 42positions the analyzer 18 outside the light path whenever the selector40 selects λ₂. When the analyzer 18 is disposed in the light path theanalyzer rotator 44 rotates the orientation of the polarization axis ofthe analyzer 18 between the first plane and the second plane. When thepolarization axis of the analyzer 18 is in the first plane, the analyzertransmits I_(P1) for subsequent intensity measurements. When thepolarization axis of the analyzer 18 is in the second plane the analyzertransmits I_(T1) for subsequent intensity measurements. When theanalyzer 18 is outside of the light path, the apparatus transmitsI_(tot2) directly for subsequent measurement.

Alternatively the orientation selection device can comprise two separateanalyzers and an analyzer selection mechanism for alternativelypositioning the first analyzer or the second analyzer in the light pathof the fluorescence emitted by the sample when the orientation selectiondevice is positioned in the light path by the positioner 42. The twoanalyzers are a first analyzer with its polarization axis in the firstplane and a second analyzer with its polarization axis in the secondplane.

In this apparatus, the wavelength selector 40 can be a single deviceinterlocked with the analyzer positioner 42 and designed to passfluorescence emissions alternately at λ₁ and λ₂. The selector 40 canalso be a pair of devices interlocked with the positioner 42, one devicefor each wavelength, the first device passing fluorescence emissions atλ₁ and the second device passing fluorescence emissions at λ₂. Theseselectors can be the emission monochromators 20 and 22 of FIG. 5.

In this apparatus, there are two measuring devices. The first measuringdevice measures only at λ₁, and the second measuring device measuresonly at λ₂. These measuring devices can comprise photodetectors. Thefirst measuring device can comprise either a single photodetector 24alternately measuring I_(P1) and I_(T1) as shown in FIG. 5, or cancomprise a pair of photodetectors measuring I_(P1) and I_(T1)separately. The second measuring device, shown as photodetector 26 inFIG. 5, measures the total intensity of the fluorescence emissions fromthe sample at λ₂, I_(tot2).

(2) Determination of I_(tot2) from Measurement of Polarized FluorescenceEmissions at "Magic Angle"

A consequence of the theory of fluorescence polarization is that whenmeasurements are made when the polarization axis of the analyzer is in aplane at an angle of 54.7°, the so-called "magic angle," from theorientation of the plane of polarization of the exciting light, theintensity of the polarized fluorescence so measured is independent ofthe degree of polarization of the emitted light. See R. D. Spencer & G.Weber, "Influence of Brownian Rotations and Energy Transfer upon theMeasurements of Fluorescence Lifetime," J. Chem. Phys. 52, 1654-1663(1970). Accordingly, the intensity of the fluorescence measured at aparticular wavelength when the polarization axis of the analyzer is atan angle of 54 7° from the orientation of the plane of polarization ofthe exciting light is directly proportional to the total intensity ofthe fluorescence emissions from the sample at that wavelength.

Therefore, the total fluorescence emission intensity at λ₂ (I_(tot2))can be determined by measurement of the polarized fluorescence intensityat the "magic angle" (I_(M2)) as long as the proportionality constant isknown. This proportionality constant can be determined by measurement ofI_(tot2) and I_(M2) for one sample, and remains constant for a giveninstrument and λ₂.

This procedure can be used regardless of the orientation of the plane ofpolarization of the exciting light and of the first and second planes ofpolarization of the emitted fluorescence in which intensity measurementsare made.

An apparatus suitable for the determination of I_(tot2) from I_(M2) isshown in FIG. 6. This apparatus has an orientation selection device forselectively transmitting plane polarized light in one of three planes:(1) a vertical plane; (2) a horizontal plane; and (3) a plane oriented54.7° from the vertical. The orientation selection device is disposed inthe path of the fluorescence emitted by the cell suspension 16. Theorientation selection device can comprise, as shown in FIG. 6, arotatable analyzer 18 always disposed in the light path of thefluorescence emitted by the cell suspension 16, and an analyzer rotator44 rotating the orientation of the polarization axis of the analyzer 18.A wavelength selector 40, shown as the pair of monochromators 20 and 22,can select either λ₁ or λ₂. The wavelength selector 40 is interlockedwith the analyzer rotator 44 so that λ₁ is selected whenever theorientation of the polarization axis of the analyzer 18 is in either thevertical plane or the horizontal plane. The selector 40 selects thesecondary wavelength λ₂ only when the orientation of the polarizationaxis is in the plane 54.7° from the vertical. The selector 40 cancomprise a single device designed to pass fluorescence emissionsalternately at λ₁ and λ₂. Alternatively, the selector 40 can comprise apair of devices each interlocked with the analyzer rotator 44. If a pairof devices is used, there is one device for each wavelength, the firstdevice selecting λ₁ and the second device selecting λ₂. These devicescan be the emission monochromators 20 and 22 of FIG. 6.

Alternatively, the orientation selection means can comprise threeseparate analyzers, one with its polarization axis in each plane, and ananalyzer selection mechanism for alternately positioning each of thethree analyzers in the path of the fluorescence emitted by the cellsuspension 16.

The apparatus further comprises a measuring device or devices formeasuring the intensities of the components of the emitted fluorescencepolarized in the three planes at the wavelength selected by thewavelength selector 40. Several arrangements of the measuring device ordevices are possible The measuring device can comprise a singlephotodetector alternately measuring I_(P1), I_(T1), and I_(M2).Alternatively, the measuring devices can also comprise two or threephotodetectors. When the wavelength selector 40 comprises two emissionmonochromators 20 and 22, as shown in FIG. 6, and the measuring devicescomprise two photodetectors 24 and 26, the photodetectors are: (1) afirst photodetector 24 disposed in the exit light path of the firstemission monochromator 20 and measuring alternately the components ofthe fluorescence transmitted by the analyzer in the vertical andhorizontal planes at λ₁ (I_(P1) and I_(T1)); and (2) a secondphotodetector 26 disposed in the exit light path of the secondmonochromator 22 and measuring the component of the fluorescencetransmitted by the analyzer 18 whenever the polarization axis of theanalyzer 18 is in the plane 54.7 ° from the vertical (I_(M2)). Themeasuring devices can also comprise three separate photodetectors. Whenthe wavelength selector 40 comprises two emission monochromators 20 and22, two of the photodetectors can be disposed in the exit light path ofthe first emission monochromator 20, the first photodetector measuringI_(P1) and the second measuring I_(T1). The third photodetector isdisposed in the exit light path of the second emission monochromator 22and measures I_(M2).

In any version of the apparatus designed to measure I_(M2) thecalculation device, such as the microprocessor 30, necessarily includescircuitry for determining I_(tot2) from I_(M2). The proportionalityconstant between I_(M2) and I_(tot2) can be determined by priormeasurements and supplied to the calculation device for storage withinthe calculation device.

EXAMPLE

Potentially SCM-responding lymphocytes were separated from samples ofperipheral blood of donors, both donors free of malignancy and donorswith cancer, in accordance with the method described in the EuropeanJournal of Cancer article by L. Cercek and B. Cercek. Samples ofperipheral blood from which the phagocytes had been removed were layeredon a Ficoll-Triosil density gradient solution having a density of 1.081g/cm³ at 25° C. and an osmolality of 0.320 Osm/kg, followed bycentrifugation at 550×g at 20° C. In accordance with the SCM procedurefor cancer testing, an aliquot of each sample of these lymphocytes wasexposed to phytohaemagglutinin (PHA) and a second aliquot of each samplewas exposed to an extract comprising pooled proteins from a variety ofmalignant tissues, referred to as cancer basic protein (CaBP). CaBP is abasic protein or group of similar basic proteins which is an effectivechallenging agent for lymphocytes from donors with cancer in the SCMtest, but causes no response in the SCM test when used as thechallenging agent for lymphocytes from donors free of cancer in the SCMtest. In accordance with our test method the ratio of the netpolarization value, P_(CaBP), for an aliquot of lymphocytes incubatedwith CaBP to the net polarization value, P_(PHA), for an aliquot oflymphocytes from the same donor incubated with PHA is an indication ofthe presence or absence of cancer in the donor. This ratio is referredto as the SCM response ratio or RR_(SCM) ; in terms of the challengingagents used:

    RR.sub.SCM =P.sub.CaBP /P.sub.PHA

This ratio has been found to be about 1.1 to about 1.8 in healthy donorsas compared to about 0.5 to about 0.95 in donors afflicted withmalignant disorders.

After incubation with either CaBP or PHA and formation of thelymphocyte-FDA suspensions in accordance with the method described inthe European Journal of Cancer article cited above, polarization valuesfor the lymphocyte suspensions with each challenging agent weredetermined by the previously published method in which the horizontallyand vertically polarized components of the fluorescence emissions weremeasured at a single emission wavelength, 510 nm, using an excitationwavelength of 470 nm. The measurements were made on a Perkin-Elmerfluorescence spectrophotometer model MPF-4.

The horizontally and vertically polarized components of the fluorescenceemissions were then measured at a second emission wavelength, 515 nm.The samples were then filtered as described in the previously publishedmethod, and the horizontally and vertically polarized components of thefluorescence emissions were then measured from the filtrate at anexcitation wavelength of 470 nm and an emission wavelength of 510 nm.

From these measurements, two separate sets of calculations were made:(1) calculations of the P_(CaBP) and P_(PHA) from the fluorescenceemissions at 510 nm from the cell suspension and the filtrate accordingto the previously published method; and (2) calculations of the P_(CaBP)and the P_(PHA) from the fluorescence emissions at 510 nm and 515 nmfrom only the cell suspension, according to the method of the presentinvention, treating 510 nm as the primary wavelength, λ₁, and 515 nm asthe secondary wavelength, λ₂. From each of these sets of calculations ofP_(CaBP) and P_(PHA), an RR_(SCM) value was determined. These resultsare shown in Table 1.

Table 1 shows that the agreement between the RRSCM as calculated by thepreviously published filtration method and as calculated by thebathochromic shift method of the present invention is quite good and iswithin acceptable limits of accuracy as predicted by an errorpropagation analysis. These results show that samples 1-5 have SCMresponse ratios of approximately 1.15 and above indicating an absence ofcancer in the donor, while the SCM response ratios for samples 6-9 areall well below 1.0 indicating that the donor is afflicted With cancer.These results obtained by the SCM test were confirmed by actualdiagnosis.

The method of measuring fluorescence polarization of the presentinvention achieves the goals which have been sought, and possesses anumber of significant advantages over the previously-describedfiltration method. The method is rapid, as all that is done is themeasurement of the polarized fluorescence emissions at two wavelengths.There is no need to measure the fluorescence polarization over anextended period of time, such as four to seven minutes, as previouslydone. The method is suitable for automation, as the fluorescencespectrophotometer can be programmed with the necessary constants forcalculation according to the method. The method requires only a smallsample. as there is no need to recover a filtrate from each sample. Themethod requires only a fluorescence spectrophotometer. Even if thespectrophotometer is not automated, it is far less difficuit to readjustthe wavelength and make two measurements on each sample than to performthe previous filtration method on each sample, and the worker performingthe measurements needs less specialized training than with the previousfiltration technique. The method is suitable for the processing of alarge number of samples. Most importantly, the method eliminates thehigh and variable backgrounds often observed with the filtration method.

Although the present invention has been described in considerable detailwith regard to certain preferred versions thereof, other versions arepossible Therefore, the spirit and scope of the appended claims shouldnot be limited to the description of the preferred versions containedtherein.

                  TABLE 1                                                         ______________________________________                                        COMPARISON OF FILTRATION AND                                                  BATHOCHROMIC SHIFT METHODS                                                    FOR DETERMINATION OF RR.sub.SCM                                                          P           RR.sub.SCM.sup.c                                       Sample                                                                              Challenging                                                                              Filtration                                                                             Shift  Filtration                                                                           Shift                                 No.   Agent      Method   Method Method Method                                ______________________________________                                        1     .sup. PHA.sup.a                                                                          0.154    0.128  1.36   1.41                                        .sup. CaBP.sup.b                                                                         0.210    0.180                                               2     PHA        0.137    0.122  1.42   1.55                                        CaBP       0.195    0.189                                               3     PHA        0.140    0.133  1.39   1.35                                        CaBP       0.195    0.189                                               4     PHA        0.163    0.161  1.28   1.14                                        CaBP       0.208    0.183                                               5     PHA        0.145    0.170  1.39   1.27                                        CaBP       0.202    0.215                                               6     PHA        0.201    0.215  0.76   0.74                                        CaBP       0.152    0.160                                               7     PHA        0.202    0.208  0.78   0.79                                        CaBP       0.158    0.164                                               8     PHA        0.202    0.196  0.80   0.84                                        CaBP       0.161    0.164                                               ______________________________________                                         Samples 1-5 were from donors free of malignancy; samples 6-8 were from        donors with cancer.                                                           .sup.a PHA is phytohaemagglutinin                                             .sup.b CaBP is cancer basic protein                                           .sup.c RR.sub.SCM is the P value for CaBP divided by the P value for PHA 

What is claimed is:
 1. A method of measuring polarized fluorescentemissions from a fluorescent material in a sample comprising thefluorescing material and a background material, the background materialcontributing background fluorescence, where the emissions spectrum ofthe background fluorescence is shifted relative to the emissionsspectrum of the fluorescing material, while compensating for thebackground fluorescence without separating the fluorescing material fromthe background material of each sample, the method comprising the stepsof:(a) exciting the sample with plane-polarized light; (b) measuring thepolarized fluorescence emissions from the sample at a primary wavelength(λ₁), the measurements being made in two planes, a first plane and asecond plane transverse to the first plane, the measurementsyielding:(i) the measured fluorescence intensity in the first plane atλ₁ (I_(P1)); and (ii) the measured fluorescence intensity in the secondplane at λ₁ (I_(T1)); (c) determining at a secondary wavelength (λ₂),different from λ₁, and within the range of wavelengths determined by themeasured shift of the fluorescence emissions spectrum due to backgroundfluorescence emissions, the total intensity of the fluorescence(I_(tot2)); (d) determining from I_(P1), I_(T1), and I_(tot2), thepolarized fluorescence emission intensity in the first plane (I_(P1B))and the second plane (I_(T1B)) emitted by the background material at λ₁; and (e) subtracting I_(P1B) from I_(P1) to obtain a first value(I_(P1F)) and subtracting I_(T1B) from I_(T1) to obtain a second value(I_(T1F)), where I_(P1F) and I_(T1F) are the emission intensities at λ₁in the first and second planes due solely to the fluorescing material.2. The method of claim 1 wherein the first plane is parallel to theplane of polarization of the exciting light.
 3. A method of measuringpolarized fluorescence emissions from a fluorescing material in a samplecomprising the fluorescing material and a background material thebackground material contributing background fluorescence, where theemission spectrum of the background fluorescence is shifted relative tothe emission spectrum of the fluorescing material, while compensatingfor the background fluorescence without separating the fluorescingmaterial from the background material of each sample, the methodcomprising the steps of:(a) exciting the sample with plane-polarizedlight; (b) measuring the polarized emissions from the sample at aprimary wavelength (λ₁) and a secondary wavelength (λ₂) different fromλ₁ and within the range of wavelengths determined by the measured shiftof the fluorescence spectrum due to background fluorescence emissions,the measurements for each wavelength being made in two planes, a firstplane and second plane transverse to the first plane, the measurementsyielding:(i) the measured fluorescence intensity in the first plane atλ₁ (I_(P1)); (ii) the measured fluorescence intensity in the secondplane at λ₂ (I_(P2)); (iii) the measured fluorescence intensity in thesecond plane at λ1 (I_(T1)); and (iv) the measured fluorescenceintensity in the second plane at λ₂ (I_(T2)); (c) determining fromI_(P1), I_(P2), I_(T1), and T_(T2) the polarized fluorescence emissionintensities in the first plane (I_(P1B)) and the second plane (I_(T1B))emitted by the background material at λ₁ ; and (d) subtracting I_(P1B)from I_(P1) to obtain a first value (I_(T1F)) and subtracting I_(T1F)from I_(T1) to obtain a second value (I_(T1F)), where I_(P1F) andI_(T1F) are the emission intensities at λ₁ in the first and secondplanes due solely to the fluorescing material.
 4. The method of claim 3wherein the first plane is parallel to the plane of polarization of theexciting light.
 5. A method of measuring polarized fluorescenceemissions from a fluorescing material in a sample comprising thefluorescing material and a background material, the background materialcontributing background fluorescence, where the emission spectrum of thebackground fluorescence is shifted relative to the emission spectrum ofthe fluorescing material, while compensating for the backgroundfluorescence without separating the fluorescing material from thebackground material of each sample, the method comprising the stepsof:(a) exciting the sample with plane-polarized light; (b) measuring thepolarized fluorescence emissions from the sample at a primary wavelength(λ₁), the measurements being made in two planes, a first plane and asecond plane transverse to the first plane, the measurementsyielding;(i) the measured fluorescence intensity in the first plane atλ₁ (I_(P1)); and (ii) the measured fluorescence intensity in the secondplane at λ₁ (I_(T1)); (c) measuring directly at a secondary wavelength(λ₂) different from λ₁ and within the range of wavelengths determined bythe measured shift of the fluorescence emission spectrum due tobackground fluorescence emissions, the total intensity of thefluorescence emissions (I_(tot2)); (d) determining from I_(P1), I_(T1),and I_(tot2) the polarized fluorescence emission intensities in thefirst plane (I_(P1B)) and the second plane (I_(T1B)) emitted by thebackground material at λ₁ ; and (e) subtracting I_(P1B) from I_(P1) toobtain a first value (I_(P1F)) and subtracting I_(T1B) from I_(T1) toobtain a second value (I_(T1F)), where I_(P1F) and I_(T1F) are theemission intensities at λ₁ in the first and second planes due solely tothe fluorescing material.
 6. The method of claim 5 wherein the firstplane is parallel to the plane of polarization of the exciting light. 7.A method of measuring polarized fluorescence emissions from afluorescing material in a sample comprising the fluorescing material anda background material, the background material contributing backgroundfluorescence, where the emission spectrum of the background fluorescenceis shifted relative to the emission spectrum of the fluorescingmaterial, while compensating for the background fluorescence withoutseparating the fluorescing material from the background material of eachsample, the method comprising the steps of:(a) exciting the sample withplane-polarized light; (b) measuring the polarized fluorescenceemissions from the sample at a primary wavelength (λ₁), the measurementsbeing made in two planes, a first plane and a second plane transverse tothe first plane, the measurements yielding:(i) the measured fluorescenceintensity in the first plane at λ₁ (I_(P1)); and (ii) the measuredfluorescence intensity in the second plane at λ₁ (I_(T1)); (c)measuringat a secondary wavelength (λ₂) different from λ₁ and within the range ofwavelengths determined by the measured shift of the fluorescenceemission spectrum due to background fluorescence emissions, and in aplane oriented 54.7° from the plane of the exciting light, thefluorescence emission intensity in the plane at λ₂ (I_(M2)), whereby thevalue of I_(M2) is proportional to the total fluorescence emissionintensity at λ₂ (I_(tot2)) regardless of the degree of polarization ofthe fluorescence emitted by the sample; (d) determining I_(tot2) fromI_(M2) ; (e) determining from I_(P1), I_(T1), and I_(tot2) the polarizedfluorescence emission intensities in the first plane (I_(P1B)), and thesecond plane (I_(T1B)) emitted by the background material at λ₂ ; and(f) subtracting I_(P1B) from I_(P1) to obtain a first value (I_(P1F))and subtracting I_(T1B) from I_(T1) to obtain a second value (I_(T1F)),where I_(P1F) and I_(T1F) are the emission intensities at λ₁ in thefirst and second planes due solely to the fluorescing material.
 8. Themethod of claim 7 wherein the first plane is parallel to the plane ofthe exciting light.
 9. The method of claim 4 wherein the first andsecond planes are orthogonal.
 10. The method of claim 6 wherein thefirst and second planes are orthogonal.
 11. The method of claim 8wherein the first and second planes are orthogonal.
 12. The method ofclaim 9 wherein the exciting plane-polarized light is verticallypolarized and the vertically and horizontally polarized fluorescenceemissions from the sample are measured.
 13. The method of claim 10wherein the exciting plane-polarized light is vertically polarized andthe vertically and horizontally polarized fluorescence emissions fromthe sample are measured.
 14. The method of claim 11 wherein the excitingplane-polarized light is vertically polarized and the vertically andhorizontally polarized fluorescence emissions from the sample aremeasured.
 15. The method of claim 1, 3, 5, or 7 wherein λ₂ is selectedso that the absolute value of ((I_(tot1F) /I_(tot1B))-(I_(tot2F)/I_(tot2B))) is maximized where I_(tot1F) and I_(tot2F) are the totalfluorescence emission intensities from the fluorescing material at λ₁and λ₂ respectively, and where I_(tot1B) and I_(tot2B) are the totalfluorescence emission intensities from the background material at λ₁ andλ₂ respectively.
 16. The method of claim 1, 3, 5, or 7 wherein thedifference between λ₁ and λ₂ is at least about 5 nm.
 17. The method ofclaim 1, 3, 5, or 7 wherein the difference between λ₁, and λ₂ is nogreater than about 15 nm.
 18. The method of claim 1, 3, 5, or 7 whereinthere is only one λ₂.
 19. The method of claim 1, 3, 5, or 7 wherein λ₁is at the maximum of the fluorescence emission spectrum of thefluorescing material.
 20. The method of claim 9, 10, or 11 furthercomprising the step of determining the net polarization value, P, of thefluorescing material from I_(P1F) and I_(T1F).
 21. The method of claim12 wherein the determination of I_(P1B) and I_(T1B) comprises:(a)obtaining from I_(P1), I_(T1), I_(P2), and I_(T2) the total fluorescenceemissions of the sample at λ₁ and λ₂, I_(tot1) and I_(tot2), inaccordance with the following relationship:

    I.sub.tot1 =I.sub.P1 +2(I.sub.Tλ ×G),

wherein I_(tot)λ is the total intensity of the fluorescence emissionsfrom the sample at the wavelength λ, with λ being equal to λ₁ or λ₂,I_(P1) and I_(T1) are either I_(P1) and I_(T1) or I_(P2) and I_(T2)depending upon the value of λ chosen, and G is a correction factor ofthe unequal transmission of the vertically and horizontally polarizedfluorescence emissions through the optical system of a fluorescencemeasuring instrument; (b) determining a factor F representing thefraction of the total intensity of the fluorescence emissions at λ₁ dueto background fluorescence by the relationship: ##EQU7## wherein: (i)K_(a) is a ratio obtained by dividing the total fluorescence emissionintensity for the background material at λ₂, I_(tot2B), by the totalfluorescence intensity for the background material at λ₁, I_(tot1B);(ii) K_(b) is a ratio obtained by dividing the total fluorescenceemission intensity for the fluorescing material at λ₂, I_(tot2), by thetotal fluorescence emission intensity for the fluorescing material atλ₁, I_(tot)λ1F, the determination of K_(a) and K_(b) requiringseparation of the fluorescing material and the background material, thedetermination of K_(a) and K_(b) being made for at least one sampleseparate from the sample on which the measurement of polarizedfluorescence emissions is made, K_(a) and K_(b) being constants for theparticular instrumentation used; and (iii) Q is the ratio of I_(tot2)divided by I_(tot2), Q varying with the relative contributions tofluorescence of the fluorescing material and the background material;and (c) determining from the factor F the values of I_(P1B) and I_(T1B)according to the relationships: ##EQU8## where P_(k) is a constantdefined by the relationship: ##EQU9## wherein I_(P1BS) and I_(T1BS) arethe vertically and horizontally polarized fluorescence emissionintensities, respectively, at λ₁, of the background material in aseparate sample from which the fluorescing material has been removed,and G is a correction factor for the unequal transmission of thevertically and the horizontally polarized fluorescence emissions throughthe optical system of a fluorescence measuring instrument.
 22. Themethod of claim 21 further comprising the steps of:(a) determining thefollowing at a second secondary wavelength, λ₃, within the range ofwavelengths determined by the shift of the fluorescence emissionspectrum due to background fluorescence emissions:(i) the vertically andhorizontally polarized fluorescence emissions from the sample I_(P3) andI_(T3) ; (ii) the total fluorescence emission intensity from thebackground material, I_(tot3B), the fluorescing material having beenremoved from the sample; and (iii) the total fluorescence emissionintensity from the fluorescing material, I_(tot3F) ; (b) determiningfrom I_(P3), I_(T3), I_(tot3B), I_(tot3F), I_(P1), I_(T1), I_(tot1B),and I_(tot1F) a second value of the factor F, F₂ ; (c) determining theaverage value, F_(av), of the factor F, from the first value of F, F₁,determined at λ₁ and λ₂, and from F₂ ; and (d) using F_(av) indetermining I_(P1B) and I_(T1B).
 23. The method of claim 1, 3, 5, or 7wherein the fluorescing material is fluorescing cells.
 24. A method fordetermining the net polarization value, P, or lymphocytes in asuspension of a fluorogenic agent precursor, where there is a backgroundof extracellular fluorescence such that the spectrum of the fluorescentemissions attributable to the extracellular background is shiftedrelative to the spectrum of the fluorescence emissions attributable tothe lymphocytes, the method comprising the steps of:(a) exciting thelymphocytes with plane-polarized light; (b) measuring the polarizedemissions from the suspension at a primary wavelength (λ₁) and asecondary wavelength (λ₂) different from λ₁ and within the range ofwavelength determined by the measured shift of the fluorescenceemissions spectrum due to background fluorescence emissions, themeasurements for each wavelength being made in two planes, a first planeparallel to the plane of polarization of the exciting light and a secondplane transverse to the first plane, the measurements yielding;(i) themeasured fluorescence intensity in the first plane at λ₁ (I_(P1)); (ii)the measured fluorescence intensity in the second plane at λ₂ (I_(P2));(iii) the measured fluorescence intensity in the second plane at λ₁(I_(T1)); and (iv) the measured fluorescence intensity in the secondplane at λ₂ (I_(T2)); (c) determining from I_(P1), I_(P2), I_(T1), andI_(T2) the polarized fluorescence emission intensities in the firstplane (I_(P1B)) and the second plane (I_(T1B)) emitted by theextracellular background material at λ₁ ; and (d) subtracting I_(P18)from I_(P1) to obtain a first value (I_(P1F)) and subtracting I_(T1B)from I_(T1) to obtain a second value (I_(T1F)), and emission intensitiesat λ₁ in the first and second planes attributable to intracellularfluorescence from the lymphocytes; and (e) determining P from I_(P1F)and I_(T1F).
 25. A method for determining the net polarization value, P,of lymphocytes in a suspension of a fluorogenic agent precursor, wherethere is a background of extracellular florescence such that thespectrum of the fluorescence emissions attributable to the extracellularbackground is shifted relative to the spectrum of the fluorescenceemissions attributable to the lymphocytes, the method comprising thesteps of:(a) exciting the lymphocytes with plane-polarized light; (b)measuring the polarized fluorescence emissions from the sample at aprimary wavelength (λ₁), the measurements being made in two planes, afirst plane parallel to the plane of polarization of the exciting lightand a second plane transverse to the first plane, the measurementsyielding:(i) the measured fluorescence intensity in the first plane atλ₁ (I_(P1)); and (ii) the measured fluorescence intensity in the secondplane at λ₁ (I_(T1)); (c) measuring at a secondary wavelength (λ₂)different from λ₁ and within the measured range of wavelengthsdetermined by the shift of emissions spectrum due to backgroundfluorescence emissions, the total intensity of the fluorescenceemissions (I_(tot2)); (d) determining from I_(P1), I_(T1), and I_(tot2)the polarized fluorescence emission intensities in the first plane(I_(P1B)) and the second plane (I_(T1B)) emitted by the extracellularbackground material at λ₁ ; (e) subtracting I_(P1B) from I_(P1) toobtain a first value (I_(P1F)) and subtracting I_(T1B) from I_(T1)obtain a second value (I_(T1F)), the emission intensities at λ₁ in thefirst and second planes attributable to intracellular fluorescence fromthe lymphocytes; and (f) determining P from I_(P1F) and I_(T1F).
 26. Amethod for determining the net polarization value, P, of lymphocytes ina suspension of a fluorogenic agent precursor, where there is abackground of extracellular fluorescence such that the spectrum of thefluorescence emissions attributable to the extracellular background isshifted relative to the spectrum of the fluorescence emissionsattributable to the lymphocytes, the method comprising the steps of:(a)exciting the lymphocytes with plane-polarized light; (b) measuring thepolarized fluorescence emissions from the suspension at a primarywavelength (λ₁), the measurements being made in two planes, a firstplane parallel to the plane of polarization of the exciting light and asecond plane transverse to the first plane, the measurementsyielding:(i) the measured fluorescence intensity in the first plane atλ₁ (I_(P1)); and (ii) the measured fluorescence intensity in the secondplane at λ₁ (I_(T1)); (c) measuring at a secondary wavelength (λ₂)different from λ₁ and within the range of wavelengths determined by themeasured shift of the fluorescence emissions spectrum due to backgroundfluorescence emissions, and in a plane oriented 54.7° from the vertical,the fluorescence emission intensity in the plane at λ₂ (I_(M2)), wherebythe value of I_(M2) is proportional to the total fluorescence emissionintensity at λ₂ (I_(tot2)), regardless of the degree of polarization ofthe fluorescence emitted by the sample; (d) determining I_(tot2) fromI_(M2) ; (e) determining from I_(P1), I_(T1), and I_(tot2) the polarizedfluorescence emission intensities in the first plane (I_(P1B)) and thesecond plane (I_(T1B)) emitted by the extracellular background materialat λ₁ ; (f) subtracting I_(P1B) from I_(P1) to obtain a first value(I_(P1F)) and subtracting I_(T1B) from I_(T1) to obtain a second value(I_(T1F)), the emission intensities at λ₁ in the first and second planesattributable to intracellular fluorescence from the lymphocytes; and (g)determining P from I_(P1F) and I_(T1F).
 27. The method of claim 24wherein the first and second planes are orthogonal.
 28. The method ofclaim 25 wherein the first and second planes are orthogonal.
 29. Themethod of claim 26 wherein the first and second planes are orthogonal.30. The method of claim 27 wherein the exciting plane-polarized light isvertically polarized and the vertically and horizontally polarizedfluorescence emissions from the suspension are measured.
 31. The methodof claim 28 wherein the exciting plane-polarized light is verticallypolarized and the vertically and horizontally polarized fluorescenceemissions from the suspension are measured.
 32. The method of claim 29wherein the exciting plane-polarized light is vertically polarized andthe vertically and horizontally polarized fluorescence emissions fromthe suspension are measured.
 33. The method of claim 24 wherein λ₂ isselected so that the absolute value of ((I_(tot1F) /I_(tot1B))-(I_(tot2F) /I_(tot2B))) is maximized, where I_(tot1F) and I_(tot2F) arethe total intracellular fluorescence emission intensities from thelymphocytes at λ₁ and λ₂ respectively, and where I_(tot1B) and I_(tot2B)are the total fluorescence emission intensities from the extracellularbackground at λ₁ and λ₂ respectively.
 34. The method of claim 25 whereinλ₂ is selected so that the absolute value of ((I_(tot1F) /I_(tot1B))-(I_(tot2F) /I_(tot2B))) is maximized, where I_(tot1F) and I_(tot2F) arethe total intracellular fluorescence emission intensities from thelymphocytes at λ₁ and λ₂ respectively, and where I_(tot1B) and I_(tot2B)are the total fluorescence emission intensities from the extracellularbackground at λ₁ and λ₂ respectively.
 35. The method of claim 26 whereinλ₂ is selected so that the absolute value of ((I_(tot1F) /I_(tot1B))-(I_(tot2F) /I_(tot2B))) is maximized, where I_(tot1F) and I_(tot2F) arethe total intracellular fluorescence emission intensities from thelymphocytes at λ₁ and _(g) 2 respectively, and where I_(tot1B) andI_(tot2B) are the total fluorescence emission intensities from theextracellular background at λ₁ and λ₂ respectively.
 36. The method ofclaim 24, 25, or 26 wherein λ₁ is at the maximum of the fluorescenceemission spectrum of the fluorescing lymphocytes.
 37. A method fordetection of the SCM response of lymphocytes and use of the response indiagnosis of the presence of a condition or disease in a body detectableby an alteration of the SCM response, the method comprising the stepsof;(a) obtaining a sample of lymphocyte-containing body fluid from thebody; (b) separating SCM-responding lymphocytes from the body fluid; (c)contacting the SCM-responding lymphocytes with an antigen derived fromor associated with the disease or condition being tested for thereby tostimulate the lymphocytes; (d) forming a suspension of the stimulatedlymphocytes and a fluorogenic agent precursor; (e) maintaining thestimulated lymphocytes in the suspension for sufficient time to allowfor the penetration of the lymphocytes by the fluorogenic agentprecursor and its intracellular enzymatic hydrolysis to a fluorogeniccompound, thereby generating stimulated fluor-containing lymphocytes;(f) exciting the stimulated fluor-containing lymphocytes withplane-polarized light, thereby causing the fluor-containing lymphocytesas well as any extracellular background fluorescent material tofluoresce; (g) polarizing the resulting fluorescent emissions in twoplanes, a first plane parallel to the plane of the excitingplane-polarized light and a second plane transverse to the first plane;(h) measuring the polarized fluorescent emissions in the two planes at aprimary wavelength λ₁ and a secondary wavelength λ₂, λ₁ and λ₂ beingchosen such that where I_(tot1F) and I_(tot2F) are the totalintracellular fluorescence emission intensities from the lymphocytes atλ₁ and λ₂ respectively, and I_(tot2F) are the total intracellularfluorescence emission intensities from the lymphocytes at λ₁ and λ₂respectively, and I_(tot1B) and I_(tot2B) are the total extracellularfluorescence emission intensities at λ₁ and λ₂ respectively, I_(tot1F)/I_(tot1B) is unequal to I_(tot2F) /I_(tot2B), whereby the intensitiesof the polarized fluorescence in the two planes at λ₁, I_(P1) in thefirst plane and I_(T1) in the second plane, and at λ₂, I_(P2) in thefirst plane and I_(T2) in the second plane, are obtained; (i)determining F, the fraction of the total intensity of the fluorescenceemissions at λ₁ due to extracellular fluorescence by using I_(P1),I_(T1), I_(P2), and I_(T2), and determining therefrom the polarizedemissions in the first plane (I_(P1B)) and the second plane (I_(T1B))due to extracellular fluorescence at λ₁ ; (j) subtracting I_(P1B) fromI_(P1) to obtain a first value (I_(P1F)) and subtracting I_(T1B) fromI_(T1) to obtain a second value (I_(T1F)) respectively, the polarizedemissions in the first and second planes at λ₁ due only to theintracellular fluorescence from the stimulated, fluor-containinglymphocytes; and (k) obtaining from I_(P1F) and I_(T1F) the SCM responseof the fluor-containing lymphocytes to the antigen derived from orassociated with the disease or condition being tested for, whereby theSCM response of the lymphocytes is an indication of the presence orabsence of the tested for disease or body condition in the body of thedonor of the lymphocytes.
 38. The method of claim 37 wherein thelymphocyte-containing body fluid is blood.
 39. The method of claim 37wherein the first and second planes are orthogonal.
 40. The method ofclaim 39 wherein the exciting plane-polarized light is verticallypolarized and the vertically and horizontally polarized fluorescenceemissions from the suspension are measured.
 41. The method of claim 37wherein λ₂ is selected so that, where I_(tot1F) and I_(tot2F) are thetotal intracellular fluorescence emission intensities from thelymphocytes at λ₁ and λ₂ respectively, and where I_(tot1B) and I_(tot2B)are the total fluorescence emission intensities from the extracellularbackground at λ₁ and λ₂ respectively, the absolute value of ((I_(tot1F)/I_(tot1B)) -(I_(tot2F) /I_(tot2B))) is maximized.
 42. The method ofclaim 37 wherein λ₁ is at the maximum of the fluorescence emissionspectrum of the fluorescing lymphocytes.
 43. The method of claim 37wherein λ₁ is at 510 nm and is selected to be between 515 nm and 520 nm.44. The method of claim 37 wherein λ₁ is 510 nm and λ₂ is selected to bebetween 515 nm and 525 nm.
 45. The method of claim 44 wherein λ₂ is 515nm.
 46. The method of claim 44 wherein λ₂ is 525 nm.
 47. The method ofclaim 37 wherein λ₁ is 527 nm and λ₂ selected to be between 532 nm and537 nm.
 48. The method of claim 37 where λ₁ is 527 nm and λ₂ is selectedto be between 532 nm and 542 nm.
 49. The method of claim 48 wherein λ₂is 532 nm.
 50. The method of claim 24, 25, 26, or 37 wherein thefluorogenic agent precursor is fluorescein diacetate.
 51. The method ofclaim 30 or 39 wherein the step of determining I_(P1B) and I_(T1B)comprises obtaining from I_(P1), I_(T1), I_(P2), and I_(T2) the totalfluorescence emissions of the sample at λ₁ and λ₂, I_(tot1) andI_(tot2), in accordance with the following relationship:

    I.sub.totλ =I.sub.P1 +2(I.sub.T1 ×G),

wherein I_(tot)λ is the total intensity of the fluorescence emissionsfrom the suspension at the wavelength λ, with λ being equal to λ₁ or λ₂,I, and I_(P1) are either I_(P1) and I_(T1) or I_(P2) and I_(T2)depending upon the value of λ chosen, and G is a correction factor forthe unequal transmission of the vertically and horizontally polarizedfluorescence emissions through the optical system of a fluorescencemeasuring instrument.
 52. The method of claim 51 wherein the step ofdetermining I_(P1B) and I_(T1B) further comprises determining a factor Frepresenting the fraction of the total intensity of the fluorescenceemissions at λ₁, due to background fluorescence by the relationship:##EQU10## wherein: (i) K_(a) is a ratio obtained by dividing the totalextracellular fluorescence emission intensity for the background at λ₂,I_(tot2B), by the total extracellular fluorescence emission intensitiesfor the background at λ₁, I_(tot1B) ;(ii) K_(b) is a ratio obtained bydividing the total intracellular fluorescence emission intensity for thelymphocytes at λ₂, I_(tot2F), by the total intracellular fluorescenceemission intensity for the lymphocytes at λ₁, I_(tot1F), thedetermination of K_(a) and K_(b) requiring separation of the lymphocytesand the extracellular background, the determination of K_(a) and K_(b)being made for at least one sample separate from the sample oflymphocytes on which the measurement of polarized fluorescence emissionsis made, K_(a) and K_(b) being constants for the particularinstrumentation and fluorogenic agent precursor used; and (iii) Q is theratio of I_(tot2) divided by I_(tot1), Q varying with the relativecontributions to fluorescence of the lymphocytes and the extracellularbackground.
 53. The method of claim 52 wherein the values of I_(P1B) andI_(T1B) are determined from the factor F according to the relationships:##EQU11## where P_(k) is a constant defined by the relationship:##EQU12## wherein I_(P1BS) and I_(T1BS) are the vertically andhorizontally polarized fluorescence emission intensities, respectively,at λ₁, of a separate solution of the fluorogenic agent precursor fromwhich the lymphocytes have been filtered, and G is a correction factorfor the unequal transmission of the vertically and the horizontallypolarized fluorescence emissions through the optical system of afluorescence measuring instrument.
 54. The method of claim 53 furthercomprising the steps of:(a) determining the following at a secondsecondary wavelength, λ₃, within the range of wavelengths determine bythe shift of the fluorescence emission spectrum due to extracellularfluorescence emissions:(i) the vertically and horizontally polarizedfluorescence emissions from the suspension, I_(P3) and I_(T3) ; (ii) thetotal extracellular fluorescence emission intensity from the background,I_(tot3B), the lymphocytes having been removed from the suspension; and(iii) the total intracellular fluorescence emission intensity from thelymphocytes, I_(tot3F) ; (b) determining from I_(P3), I_(T3), I_(tot3B),I_(tot3F), I_(P1), I_(T1), I_(tot1B), and I_(tot1F), a second value ofthe factor F, F₂ ; (c) determining the average value of the factor F,F_(av), from the first value of F determined at λ₁ and λ₂, F₁, and fromF₂ ;and (d) using F_(av) in determining I_(P1B) and I_(T1B).